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V515 CLASS NOTES
FALL 2003
DR. BONANNO
1
1. WATER, PH AND BUFFERING 4
2. CARBOHYDRATES 8
3. NUCLEOSIDES, NUCLEOTIDES, NUCLEIC ACIDS, DNA AND RNA 12
4. LIPIDS 18
5. AMINO ACIDS AND PROTEINS 22
6. HEMOGLOBIN 30
7. ENZYMES 35
8. REGULATION OF ENZYME ACTIVITY 40
9. CELL STRUCTURE AND FUNCTION 47
10. MEMBRANES 52
11. SIGNAL TRANSDUCTION 55
12. GLYCOLYSIS, CITRIC ACID CYCLE & OXIDATIVE PHOSPHORYLATION 59
13. GLYCOGEN METABOLISM 67
14. GLUCONEOGENESIS 71
15. FATTY ACID CATABOLISM 74
16. LIPID SYNTHESIS 78
17. REVIEW OF HORMONE ACTION 85
18. AMINO ACID METABOLISM : UREA FORMATION CHAPTER 17 IN BOOK 92
19. PURINES, PYRIMIDINES AND HEME 97
20. GENOME ORGANIZATION 102
21. DNA REPLICATION 106
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22. DNA REPAIR AND RECOMBINATION 109
23. RNA SYNTHESIS 115
24. RNA PROCESSING 117
25. PROTEIN SYNTHESIS 121
26. PROTEIN TARGETING AND TURNOVER 125
27. BIOTECHNOLOGY 129
28. GENE REGULATION IN PROKARYOTES 134
29. ANIMAL VIRUSES 143
30. ONCOGENES AND HUMAN CANCER 148
31. THE CRYSTALLINE LENS 153
32. COLLAGEN 157
33. PHOTORECEPTOR BIOCHEMISTRY 158
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Water V515 Dr. J. Bonanno
1. Water, pH and Buffering
A) Water as Solvent
Water is the predominant solvent for biological materials. Water forms a dipole: i.e., the charge
distribution is skewed. The oxygen atom is electron rich while the hydrogen nuclei are areas of
positive charge. Thus it is called a polar solvent and will help solubilize polar solutes.
O2-
+
H H+
Water forms hydrogen bonds with itself and other polar molecules. Definition: The electrostatic
interaction between the hydrogen nucleus on one water molecule and the unshared electron pair
of another is termed a hydrogen bond.
Molecules that can form hydrogen bonds with water are readily solvated. These include alcohols
(-OH), sulfhydryl groups (-SH), amines (NH2), and carbonyl groups (C=O, includes esters,
aldehydes and ketones).
Long chain hydrocarbons cannot form hydrogen bonds and are therefore insoluble in water.
They form droplets in water to minimize the surface interaction with water molecules.
B) Water Dissociates
H2 O + H2 O ↔ H3+ O + OH −
Hydronium ion usually written as H+.
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Water V515 Dr. J. Bonanno
Some Definitions:
From Chemistry we know that the concentrations of products and reactants can be related to an
Equilibrium constant, K, sometimes called the dissociation constant (KD) or for the reverse
reaction the association constant (KA), where for the above reaction,
[ H + ][OH − ]
KD =
[ H 2 O]
What is the concentration of H+ and OH- in pure water?
KD = 1.8 x 10-16
So, what is the molarity of water?
The ion product, Kw = [H+][OH-] = 10-14 for any solution.
So if you know [H+], you can calculate [OH-] or vice versa.
C) pH is the negative Log of the [H+]
DEFINITION: pH = -log[H+],
so the pH of pure water is –log (1 x 10-7) or 7.
What is the pH of a solution whose [H+] = 3.2 x 10-4?
pH = –log (3.2 x 10-4)
= -log (3.2) – log (10-4)
= -0.5 + 4
= 3.5.
What about a solution that has 4 x10-4 mol/L of hydroxide (OH-) ion?
[H+] = 10-14/4x10-4
= 2.5 x 10-11.
pH = -log(2.5x10-11)
= 10.6
Acids are proton donors and bases are proton acceptors. Strong acids (e.g. HCl) or bases (e.g,
NaOH) completely dissociate and weak acids (acetic) or bases (e.g., NH4OH) partially
dissociate.
The amount of acid (or base) produced by a weak acid (or base) depends on its dissociation
constant, KD.
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Water V515 Dr. J. Bonanno
For a generic acid, HA is the acid, and A- is the conjugate salt:
HA ↔ H + + A −
Example: What is the pH of a 0.1 M solution of acetic acid?
KD = 1.76x10-5 for acetic acid.
KD x [HAc] = [H+][Ac-]
And [HAc] = 0.1 M.
For every HAc molecule that dissociates one H+ and one Ac- is formed. Therefore, K x [HAc] =
[H+]2. Therefore
[ H + ] = K d [ HAc]
So for a 0.1 molar solution of acetic acid [H+] = 1.33x10-3 and pH = 2.87.
For weak acids and bases K is represented by pK, where pK = -log K.
So the pK of acetic acid is –log (1.76x10-5) = 4.75.
When the unprotonated [A-] and protonated [HA] form of the acid are equal, then [H+] = K or
similarly, pK = pH. Therefore the pK of an acid group is that pH at which the protonated and
unprotonated species are present at equal concentrations. Buffering (see below) is best at the pK.
The Henderson-Hasselbach equation (memorize) can be used to predict the pH of a solution or
the relative amounts of protonated and unprotonated species.
[ A− ]
pH = pK + log
[ HA]
For example if [HA] ~ 0.1 molar for a weak acid, [A-] = 1.33x10-3 molar and pK is 4.75 then pH
is 2.87.
AS we said above, when [A-] = [HA] then pH = pK.
The charge on many reactive groups can affect biological activity of many proteins, so they can
be very sensitive to pH.
D) Buffers
A buffer is a weak acid (or base) and its conjugate base (or acid) that cause solutions to resist
changes in pH when an acid or base is added.
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Water V515 Dr. J. Bonanno
Effectiveness of a buffer depends on its concentration and its pK relative to the pH of the
solution. A buffer works best within one pH unit of its pK.
Without buffers, adding a small amount of acid to a system (e.g., exercising produces acid)
would change the pH drastically.
Example: Solution of acetate. If 0.1 equivalents of base are added to an acetate solution at pH
4.8, the pH will go up to ~4.85. If the solution were unbuffered, the pH would be ~13! If 0.1
equivalents of acid are added to an acetate solution at pH 4.8, the pH will drop to ~4.75.
The primary inorganic buffer in the body is phosphate ~1-2 mM. Proteins can also act as buffers
since their amino acid components can have pKs near 7. However, the primary organic buffer is
CO2/HCO3-, present at ~25 mM. Thus the CO2/HCO3- buffering system is the major buffer
system of the body.
CO2 + H2 O ←⎯→ H2 CO3 ↔ HCO3− + H +
CA
CA is an enzyme called carbonic anhydrase that makes the hydration and dehydration of CO2
very fast.
CO2 is produced in the tissues and is expelled in the lungs, so there is no net acid produced by
CO2. However, HCO3- can act to buffer other acids, e.g. lactic acid.
Respiratory acidosis occurs when breathing is impaired. Why?
Respiratory alkalosis occurs during hyperventilation. Why?
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Sugars_2 V515 J. Bonanno
2. CARBOHYDRATES are short chain hydrocarbons with hydroxyl groups and an aldehyde or
ketone group (see figure).
Nomenclature: Hexoses (6 carbons); Pentoses (5 carbons); Aldose(aldehyde); Ketose (ketone);
Stereoisomers (same formula, but differ in position of OH on one or more of their asymmetric
carbons);
D and L isomerism: D- refers to OH group written to the right on asymmetric carbon #5, L on
the left. The mirror image of D-glucose is L-glucose. Only D-glucose can be metabolized.
Epimers of glucose (differ by only 1 position on carbon #2, 3 or 4); Thus galactose, glucose, and
mannose are all epimers. Fructose is an example of Aldose-ketose isomerism.
B) Most sugars exist in solution as ring structures where the carbonyl group has reacted with a
hydroxyl group.
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Sugars_2 V515 J. Bonanno
Hexoses are the major energy supply to the body.
Pentoses are 5-carbon sugars. D-Ribose is a 5-carbon aldose sugar that joins with purines and
pyrimidines to form nucleic acids.
C) Other types of Sugars:
Amino Sugars: substitute an NH3+ for a –OH, e.g. glucosamine has amine on #2 carbon.
Acid sugar: substitue carboxylic acid at carbon 1 or 6, e.g. glucuronate.
Sulfated sugars: sulfate on aldehyde or amino groups of glucosamine.
Phosphorylated sugars: phosphate on #6 of glucose is glucose 6-phosphate. It’s charged and
helps keep sugar in the cell.
Deoxy sugars: missing –OH, e.g. 2-deoxyribose This is the sugar in the DNA backbone.
Polyol: aldehyde reduced to –OH, e.g. sorbitol
D) Glycosides (Have a glycosidic bond)
1) Disaccharide
2) Oligosaccharides 3 to 12 sugars
3) Polysaccharides many thousands in chains and branched, e.g.
glycogen
4) Cardiac Glycosides, e.g. Digitalis
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Sugars_2 V515 J. Bonanno
Glycosaminoglycans (mucopolysaccharide) contains amino sugars and acid sugars, e.g.
hyaluronic acid, chondroitin sulfate and heparin.
When connected to proteins they are called Proteoglycans. Function: packing substance,
hydrophilic, lubricating, and cushioning. Components of connective tissue.
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Sugars_2 V515 J. Bonanno
Glycoproteins: membrane proteins with oligo- or polysaccharides. Sugars on membrane protein
create a charged surface that is more hydrophilic than bare phospho-lipid membrane. Some
glycoproteins act as surface receptors , e.g. blood group antigens, major histocompatibility
proteins.
Glycogen is a major polysaccharide in the body. It is the major storage form of glucose found in
every cell but predominant in the liver and muscle.
Glycogen Linkage:
Cardiac Glycosides = Sugar + Steroid; Below is Ouabain (related to Digitalis):
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Nucleic Acids_3 V515 J. Bonanno
3. Nucleosides, nucleotides, Nucleic acids, DNA and RNA
Below are 4 ribonucleosides. They contain a sugar and a base.
Sugar
Ribose (a 5 carbon sugar) or Deoxyribose missing hydroxyl on 2’ carbon. Come from Pentose
Phosphate Shunt (Chapter 12 of Notes).
Bases (Two types)
Purines: Adenine and guanine .
Pyrimidines: cytosine, uracil (RNA); thymine (DNA).
Can be synthesized de novo. No dietary intake, made in the liver.
Bases connected through a nitrogen to #1 carbon on sugar (N-linked glycosidic bond).
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Nucleic Acids_3 V515 J. Bonanno
Nucleotides contain phosphate group(s)
There can be 1, 2 or 3 phosphates on carbon #5, e.g. AMP, ADP, ATP.
ATP is the major energy carrier that is used in the body. ADP is its breakdown product.
Ratios of ADP/ATP are an index of the energy status of the cell. GTP can also serve as an
energy source for a few metabolic reactions. GTP is also an important substrate in Signal
Transduction (chapter 11 of Notes)
A Physiologic Aside:
Purinergic receptors
All of these nucleotides and the nucleoside adenosine can serve as signaling molecules in nerves
or paracrine signals in epithelia or muscle. For example, ATP is released when cells are
damaged. EcoATPases on the cell membrane can break them down to ADP, AMP and
adenosine. This can change the physiology of adjacent cells in order to respond to the injury.
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Nucleic Acids_3 V515 J. Bonanno
Nucleic acids (DNA and RNA) are made up of single or double-stranded chains of nucleotides.
Each nucleotide is made up of a sugar (ribose or deoxyribose) , a base (purine or pyrimidine)
and a phosphate group.
For DNA and RNA the phosphate group is linked to the 5’ carbon and 3’ carbon. Gives
directionality to polymer. 5’ free end on left and ending with 3’ free is called: 5’ to 3’.Sequence
of nucleotides provides the genetic code.
SINGLE-STRANDED DNA:
5’
3’
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Nucleic Acids_3 V515 J. Bonanno
Secondary Structure (Double-stranded DNA)
Bases are hydrophobic. Sugars and phosphates are hydrophilic. So bases stack to hide from
aqueous solvent. They form hydrogen bonds with each other, forming complementary base
pairs. A-T(U) and G-C. So bases are on the inside of the double helix and phosphates are on the
outside.
DNA is almost always base paired, but RNA is only sometimes base paired. DNA is double-
stranded, RNA is single-stranded.
Major points
1) sugar & phosphates (“backbone”) on the outside to interact with the solvent.
2) One strand runs 5’ to 3’ left to right on the page, the other runs 3’ to 5’ left to right.
Write 5’-ATTCGT-3’ complementary strand would be:
3’-TAAGCA-5’
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Nucleic Acids_3 V515 J. Bonanno
B. DNA Tertiary Structure
DNA seconday structure is a double-helix. This double helix can be wound into different shapes
as well (called tertiary structure).
Tertiary structure is provided by proteins that bind DNA. The binding of proteins depends on the
sequence of local stretches of DNA. Thus tertiary structure of DNA is determined by its
sequence. Tertiary structure determines availability of genes for transcription. The major
structural protein class are the histones. Histones are very basic (lots of + charged amino acids),
so they can bind to (-) charged phosphates on backbone.
Bacterial DNA is circular. Eukaryotic DNA is linear, but separated into domains. DNA is
anchored every 50-100kb on a protein matrix.
DNA can have additional twists or “supercoiling”. Caused by bound proteins, e.g. DNA or RNA
polymerase. Topoisomerases can untwist DNA by making single or double-stranded cuts.
Important for replication, transcription and “untangling” for mitosis.
C. DNA Denaturation
Denature by raising pH or temperature or lowering ionic strength. Yields two single-strands.
Need more adverse conditions to break a strand. “Melting” point of double-stranded DNA is
dependent on ionic strength, pH and GC content (has 3 hydrogen bonds).
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Nucleic Acids_3 V515 J. Bonanno
D. HYBRIDIZATION
Powerful technique for detecting particular DNA or RNA sequences. (Southern, Northern, PCR).
Southern Blotting:
(1) DNA subjected to electrophoresis. Since DNA has uniform charge, it moves by size.
(2) Transfer single strands to a nitrocellulose filter
(3) DNA is denatured
(4) add a complementary sequence probe that is tagged. See Figure 2.14 in book.
Northern blotting is the same, but for RNA. However, the RNA is always denatured.
Western blotting is for protein interactions with antibodies.
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Lipids_4 V515 J. Bonanno
4. Lipids
a) Fatty acids: straight aliphatic chains with a methyl group at one end and a carboxylic acid
group at the other end.
Saturated or fully hydrogenated fatty acids have all single bonds. Monounsaturated indicates
one double bond and polyunsaturated indicates two or more double bonds. Most naturally
occurring double bonds in fatty acids are in the cis configuration.
The melting point of fatty acids increases with chain length and decreases with the degree of
unsaturation. Fluidity of cell membranes is partially determined by fatty acid content.
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Lipids_4 V515 J. Bonanno
b) Acylglycerols
The carboxylic acid group of fatty acids can react with alcohol (hydroxyl) groups to form esters.
The three carbon alcohol, glycerol, can react with fatty acids to form mono-, di-, or
triacylglycerols (sometimes called triglycerides).
c) Phosphoacylglycerols: Carbon 1 and 2 are esterified to fatty acids and carbon 3 has a
phosphoryl group, e.g. phosphatidic acid. Major substituted phosphoacylglycerols are the major
components of the cellular membrane.
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Lipids_4 V515 J. Bonanno
d) Eicosanoids
These compounds are derived from arachadonic acid, a 20 carbon polyunsaturated fatty acid.
Eicosanoids include prostaglandins, leukotrienes and thromboxanes. They have hormone like
function and are also mediators of inflammation.
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Lipids_4 V515 J. Bonanno
f) Steroids
Four ring structure called the steroid nucleus. Cholesterol is synthesized only in animals, not
plants. Cholesterol is the parent compound from which other steroids are produced. The
hydroxyl group on cholesterol will react with fatty acids to form an ester. These cholesterol
esters are very insoluble in blood and are stored as droplets associated with lipoproteins.
Bile salts (cholic acid) are amphipathic (hydrophilic and hydrophobic portions exist) and serve to
emulsify lipids in the gastrointestinal tract.
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Amino Acids-Proteins_5 V515
5. Amino Acids and Proteins
Amino acids are small molecules that function as: 1) building blocks of proteins, 2)
neurotransmitters, 3) components of small peptide hormones
Amino acid functional groups
a) carboxyl and amino groups
b) R-group or side chain (determines functionality in proteins)
At physiological pH, the amino group carries a proton and is positive charged and the carboxylic
acid group has dissociated a proton and is negatively charged.
Major properties to consider are electrostatic charge, hydrophobicity and polarity.
Electrostatic charge
Net charge is determined from the side chain charge in addition to α-carboxylic and amino
groups. At physiological pH, acidic side chains are negatively charged (e.g., glutamic acid,
(glutamate is the salt) and aspartate, are weak acids); basic side chains are positively charged
(lysine, arginine, histidine). The strength of the charge is reflected in its pK and the ambient pH.
The pK can be altered by the protein environment.
Hydrophobicity
Aliphatic and aromatic groups make amino acids hydrophobic. These groups interact with each
other and other hydrophobic molecules, e.g. lipids. These groups do not interact with polar or
charged amino acids or water.
Polarity
Polar groups that can form hydrogen bonds and charged groups (which are also polar) are
hydrophilic and as such will associate with water or other polar groups.
Weakly hydrophobic due to small size: glycine, cysteine and alanine.
Strongly hydrophobic: leucine, isoleucine, valine, phenylalanine, proline and methionine.
Amphipathic: tyrosine and tryptophan. Lysine and arginine are charged, but have hydrophobic
linkers.
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Amino Acids-Proteins_5 V515
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Amino Acids-Proteins_5 V515
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Amino Acids-Proteins_5 V515
Titration of amino acids
− +
R − COOH ↔ R − COO + H pK ~ 2 Acid: proton donor
+ +
R − NH 3 ↔ R − NH 2 + H pK ~ 9-10 base proton acceptor
The pK of a particular group is the pH at which it is half ionized.
The pI or isoelectric point is the pH at which the entire molecule has zero net charge.
Glycine
Histidine
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Amino Acids-Proteins_5 V515
Peptide Bond
Amino acids can be linked by a peptide bond. α-carboxyl and amino join and water is
eliminated.
Peptide bond is very stable and very hard to hydrolyze. However, enzymes called proteases are
present in the body that can break peptides more easily. Peptides are less than 10 amino acids.
The atoms in a peptide bond cannot rotate. However rotation can occur on the adjacent α-
carbon. This can lead to many possible shapes and configurations.
Proteins (> 10 amino acids)
Functions: Structural (collagen, elastin), mechanical (actin/myosin), enzymes, transporters,
hormones, receptors
Components: 20 amino acids strung in a polymer or “polypeptide”. Structure defines the activity
and structure is determined by the sequence of amino acids.
Protein Structure
1) Primary structure is the sequence of amino acids.
2) Secondary structure or local folding: rotations of chains around α-carbon. Amount of
rotation is determined by rotability of α-carbon, e.g. proline can’t rotate, and side chain
interactions, e.g. electrostatic, hydrogen bonding, disulphide bonding and hydrophobic
interactions.
Globular proteins have short uniform structure lengths. There are two common uniform
secondary structures the α-helix and the β-sheet.
α-helix: The C=O and N-H residues of the peptide bond are hydrogen bonded to each other 4
residues apart, makes it very stable. R-groups are on the outside of the helix. It is possible that
one side of the helix will be hydrophilic and the other side hydrophobic (i.e., amphipathic). The
hydrophobic side would associate with other hydrophobic amino acids (or lipids) and the
hydrophilic side would associate with water of other hydrophilic amino acids.
Each amino acid has a different probability of being found in an α-helix. For example proline is
a helix breaker because it cannot rotate.
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Amino Acids-Proteins_5 V515
β-sheet : The C=O and N-H residues of the peptide bond are hydrogen bonded to each other, as
in α-helix, but on different chains or the same chain looped back. This gives a flattened
structure.
α-Helix
β-pleated sheet
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Amino Acids-Proteins_5 V515
Reverse or Hairpin turns: Amino acids like proline that cause chain to turn and fold back on
itself, leading to a globular structure.
Random Coil: None of the above. Bonds can rotate easily, little if any hydrogen bonding.
Often bound to something else, e.g. histones in chromatin.
Functional Motifs and Domains: Motif is a short section (<25 a.a.) with identified function.
For example, Ser/Thr-Pro can be phosphorylated on Ser or Thr by proline –directed kinases, e.g.,
Protein Kinase A. A domain is larger (>50 a.a.). An example of a domain would be the
catalytic portion of an enzyme.
Structural Domains: Large stretches of a.a. that are conserved for a particular type of structure,
e.g. membrane proteins have three domains, membrane spanning domain (α-helix), extracellular
domain (binds long chain sugars) and intracellular domain.
Uniform structures are formed by repeating sequences typically found in structural proteins, e.g.
Collagen.
3) Tertiary Structure: Overall three-dimensional structure of a protein.
4) Quaternary Structure: Two or more polypeptide chains united by non-covalent bonds and
disulfide bridges. Subunits could have different functions, e.g. inhibitory or catalytic or may
involve cooperative binding, e.g. hemoglobin.
Post-translational modification: Phosphorylation (cytoplasm), addition of sugars (glycosylation
occurs primarily in ER and Golgi), hydroxylation (collagen, need vitamin C).
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Amino Acids-Proteins_5 V515
Protein Purification
Use charge (ion-exchange), hydrophobicity (reverse-phase solvent elution), size
(molecular sieves) and functional affinity (matrix contains binding group) to separate out
proteins. These processes often preserve function, done in the cold.
Final Purification and Identification of Proteins usually uses denatured proteins. Denature by
heat, detergents and reduction: Gel Electrophoresis, SDS (size only) . Isoelectric focusing:
Separate first in pH gradient. Each protein moves in electric field until it reaches a pH equal to
its pI. Combine with SDS-electrophoresis.
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Hemoglobin_6 V515
6. Hemoglobin
Function: Carry oxygen from the lungs to the tissues, release oxygen, accept CO2 and H+ and
deliver to the lungs.
Associated inherited disorders: Sickle Cell Anemia, thalassemias
Environmental factors: exercise, high altitude, carbon monoxide poisoning.
Heme
Prosthetic group (nonpolypeptide part), contains reduced iron (ferrous) (Fe2+) in a hydrophobic
porphyrin ring that interacts with hydrophobic amino acids. Most of the polar residues are on the
surface.
Myoglobin (the holoprotein) contains Heme, but only one polypeptide chain or apoprotein called
globin. Compact spherical structure with a deep hydrophobic cleft where Heme is located. Two
histidine groups (one covalently bound) interact with the Fe2+ and affect the affinity of oxygen
binding. What’s the pK of histidine R group?
Myoglobin takes up O2 that has diffused from blood. It then shuttles it to the mitochondria in the
muscle cell. Myoglobin acts as an O2 reserve for quick action.
MbO2 ↔ Mb+O2, so KD=[Mb][O2]/[MbO2]
Thus ratio of oxyMb to deoxyMb = [MbO2]/[Mb] = [O2]/KD
Or fractional (%) saturation =[MbO2]/([Mb] + [MbO2])
= [O2]/([ O2]+K), K (or P0.5)= 2.75 torr
gives hyperbolic function. This low KD indicates high affinity for oxygen. Gives up oxygen in
the cell when needed by mitochondria which operate well at <2 torr.
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Hemoglobin_6 V515
Hemoglobin
Apparent solubility of oxygen is increased by hemoglobin.
Quaternary Structure
4 polypeptides, 2 α and 2 β subunits, i.e. can bind 4 oxygens.
There are two general conformations of hemoglobin. The R or relaxed structure binds oxygen
more readily, the T or taut structure releases oxygen more readily. Strength of electrostatic
interactions among the chains governs tightness.
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Hemoglobin_6 V515
Completely deoxygenated hemoglobin is in a T structure and has difficulty binding oxygen. As
oxygen binds the structure relaxes and allows additional oxygen to bind more easily.
Quaternary structure provides a binding site for 2,3-bisphosphoglycerate (BPG) and an area for
interaction between the two β−subunits.
BPG is a highly charged molecule that can bind between the two β-chains through electrostatic
bonds and stabilizes hemoglobin into the T structure. BPG is made from an intermediate
glycolytic compound. BPG is low in most cells, but high in blood.
At high altitude or during exercise, [BPG] increases as does blood flow and the number of RBCs.
So BPG, which would be higher during metabolic demand helps release oxygen to the tissues.
This is the same as saying that BPG shifts the saturation curve to the right.
CO2 and H+, which are higher during metabolic demand also maintain the T structure and thus
help release oxygen.
In contrast to myoglobin, oxygen release by hemoglobin begins over the range 30-40 torr, P0.5
=27 torr.
Because of four binding sites and cooperativity between subunits, hemoglobin has a more
complex binding curve. Binding of oxygen to one subunit enhances binding to the next, called
positive cooperativity.
HILL EQUATION FOR BINDING of Oxygen: log([HbO2]/[Hb]) = (n)log[O2]-logK
The Hill coefficient (n) gives a measure of cooperativity. A Hill coefficient = 4 would indicate
complete cooperativity for hemoglobin.
For Myoglobin n =1, gives hyperbolic function. For Hemoglobin n =2.8, gives sigmoidal
function.
Picture of cooperativity: 1) Oxygen binding to deoxy form relaxes Fe2+ into the plane of the
porphyrin ring. 2) Relaxation is transferred to the polypeptide causing breakage of salt links
between all four chains, squeezing out BPG and thus enhancing further oxygen binding. This is
a T to R transition.
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Hemoglobin_6 V515
Bohr Effect (The effect of pH on oxygen affinity)
Protons favor the formation of salt bridges (Taut) by binding to Histidine residues. Thus at low
pH, oxygen is released more easily.
Affect of CO2
CO2 binds directly to terminal α-amino group of hemoglobin:
R-NH2 + CO2 ↔ R-NHCOO- +H+ (Carbamate favors salt bridge between α and β chains, i.e.
taut; protons bind histidines). Thus hemoglobin acts as a pH buffer, as well. 15% of CO2 in the
blood is carried by hemoglobin.
At the lungs the reverse reaction occurs. Oxygen promotes proton release.
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Hemoglobin_6 V515
Normal Hemoglobins
Hb A is α2β2. Hb A1c is glucosylated. Used as a long term measure for plasma glucose levels.
Hb F is α2γ2 which has higher affinity in order to deliver oxygen from maternal blood to fetus.
Disease
A) Gene Deletion
α-Thalassemia (absence of gene) (homozygous, two α-chain genes per chromosome, i.e. four
alleles defective) no α-chains, thus no Hb F or Hb A. Fetus makes a hybrid, e.g. β4, from other
globins, but dies at or near birth.
Heterozygous individuals have 3, 2, (no symptoms) or 1 α-gene (mild symptoms).
β-thalassemia (no β-chains, only one gene per chromosome) can make hybrids and have more
Hb F.
B) Mutations
Hemoglobinopathies due to mutations in α or β chains.
Hb S (Sickle Cell) glu6 changed to val. So polar changed to hydrophobic. Reduced solubility
more aggregation. Occurs predominately in deoxy form. So oxyhemoglobin is OK.
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Enzymes_7 V515
7. ENZYMES
Enzymes are typically Proteins. Some coenzymes (explained later) are small organic molecules
that sometimes contain inorganic molecules. More recently it has been found that RNA can also
have enzymatic activity (Ribozymes).
Properties of enzymes:
1) Catalysts, make reactions go faster
2) very specific action (e.g. a.a. sequence, specific glycosidic bonds,etc.)
3) can be regulated (allosteric, feedback, gene regulation)
4) localized to specific cell structures (e.g. lysosomes)
KINETICS (Speed) vs. Thermodynamics (Yes or no)
Enzymes make reactions go faster. They do not change the equilibrium, i.e. Keq is
unchanged!
Enzymes work by lowering the energy content of intermediate reaction steps, i.e. lower
activation energy. They do not affect the net energy change (∆Gtotal) is unaffected!
∆Gformation ∆Gdecay
⎯ ⎯
A + B ←⎯⎯⎯ → AB ←⎯⎯ → 2C
In this reaction, an enzyme would lower the ∆G formation (which is +), this would have the
effect of making the reaction go much faster. Without the enzyme the reaction would still go
from left to right because the net ∆G is negative, but it could be very slow.
Overall Rates affected by:
1) temperature
2) pH, changes charge on enzyme or substrate
3) [substrate]
4) [enzyme]
5) inhibitors
K1 is the forward reaction rate constant and K-1 is the
nA + mB ← ⎯⎯→ A nB m
K 1, K − 1
reverse reaction rate constant.
Rate1 = K 1[ A ]n [ B ]m Rate constants and equilibrium constants describe
Rate−1 = K −1[ AB ] different things, but they are related.
Equilibrium: Rate−1 = Rate1
Since Keq is unaffected by enzymes, the rate
[ AB ] K1 constants must increase by the same factor or
K eq = = enzymes must change the effective concentration of
[ A ]n [ B ]m K − 1 the substrates.
∆ G 0 ' = − RT ln K eq
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Enzymes_7 V515
In practice, enzyme catalyzed reactions are quantitated by measuring initial velocities (moles of
product formed/time). At later times the reaction will slow due to: substrate being used,
product accumulation, loss of activity (denaturation) of the enzyme.
[Product] vs. time
Initial Velocity (Vi) vs. [S] is a hyperbola for most reactions.
V max[ S ]
This is described by the Michaelis-Menton equation: Vi =
Km + [ S ]
a) at[S] = Km, Vi = ½ Vmax
b) at [S] = .1Km Vi~Vmax[S]/Km
c) at [S]>>Km Vi = Vmax[S]/[S] = Vmax.
Lineweaver-Burk Plot:
1 Km 1 1
Rearrange: = +
Vi Vmax [ S ] V max
Slope = Km/Vmax,
At 1/[S] = 0, 1/V= 1/Vmax
At 1/V = 0, 1/[S] = -1/Km
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Enzymes_7 V515
Meaning of Vmax and Km
Vmax is related to the enzyme concentration and turnover number of enzyme:
Vmax = Kcat x [Enz].
Km is independent of [Enz] and approximates 1/affinity or dissociation constant of Enzyme with
substrate. Thus a low Km is a tight binding (i.e., high affinity).
Most phsyiological substrate concentrations are near the Km for the reaction. This gives best
control when [S] changes. If Km is very low compared to [S], (e.g. hexokinase) then reaction
rate is independent of [S].
Isozymes
Isozymes are physically distinct forms (varying amino acid sequence) of an enzyme that catalyze
the same reaction. Isozymes have different Km values to suit the particular needs of the tissue.
They can be used in medical diagnosis following tissue injury. Example LDH (H and M types)
tetramer (4 subunits). H3M is typical in serum, but H4 increases after heart attack. M4 is found
in muscle and liver. So M4 in serum could indicate liver disease. Five possible combinations:
H4, H3M1, H2M2, H1M3, M4.
M4 M3H1 M2H2 M1H3 H4
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Enzymes_7 V515
Enzymes are used in Serum Assays used Clinically
Example for lactate determination:
lactate +NAD→pyruvate +NADH.
Use standard amounts of LDH and NAD, assay NADH formation
NADH absorbs at 340nm.
There are numerous assays for glucose. These are important in testing for diabetes or other
metabolic disorders. Here is one example:
Glucose Oxidase
Glucose + O2 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ Gluconate + H 2O2
Peroxidase
H2O2 + Dye ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ Dye *
The dye is quantitated by a colorimetric assay using a spectrophotometer.
Cofactors are non-protein molecules that participate in the catalytic process. They include trace
elements, zinc, copper, Mg (e.g., MgATP), Fe, and specific organic coenzymes like pyridoxal
phosphate, which is derived from B6 vitamins, and NAD. Body can’t synthesize some, these are
therefore essential in the diet.
Coenzymes are organic cofactors. Enzyme activity often requires Coenzymes (tightly bound =
prosthetic group, not bound = secondary substrate) a non-polypeptide part of the enzyme, (e.g.
HEME is a covalent coenzyme or prosthetic group).
Oxidation-reduction reactions, very commonly use vitamin coenzymes, like NAD
(nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). These act as
secondary substrates. In reduction reactions, the substrate loses two hydrogens (electrons) and is
therefore oxidized. The two electrons are transferred to oxidized NAD+ giving reduced NAD+ or
NADH. One H is lost to the solvent.
A Vitamin is an organic molecule required for certain metabolic functions that must be supplied
in very small amounts (less than 50 mg/day) because we either cannot synthesize it or cannot
synthesize enough to meet our needs.
Minimum daily requirement (MDR) enough to prevent signs and symptoms of disease, however
biochemical abnormalities could still be present. The recommended daily allowance (RDA)
should avoid biochemical deficiencies.
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Enzymes_7 V515
Water Soluble Vitamins
This includes all B vitamins and vitamin C.
Thiamin (B1) needed for maximum utlization of sugars for energy.
Nutritional deficiency gives symptoms of beri-beri (peripheral neuropathy) or Wernicke’s
Encephalopathy: confusion, ataxia and ophthalmoplegia, often due to alcoholism.
Riboflavin (B2) forms FMN (riboflavin 5’-phosphate), which reacts with ATP to form FAD.
Used in many reactions, especially oxidative phosphorylation in mitochondria. Deficiency cause
angular stomatitis (fissures at the corners of the mouth) and breakdown of skin.
Niacin (nicotinic acid) is a precursor to NAD+. Oxidation-reduction, oxidative phosphorylation.
Deficiency is called Pellagra (dermatitis, diarrhea and dementia).
Pantothenic acid (a B vitamin) forms Coenzyme A. Deficiency is rare.
Pyridoxal Phosphate (B6) needed in amino acid metabolism. Deficiencies most often seen during
pregnancy and alcoholism (anemia, neuropathy)
Biotin (a B vitamin) From diet and intestinal bacteria, so deficiency is rare. Needed for
carboxylase reactions. e.g. hemoglobin.
Tetrahydrofolate (folic acid, a B vitamin) Needed for purine synthesis. Deficiency causes
anemia, heart disease.
Cyanocobalamin (vitamin B12) intestinal absorption requires intrinsic factor. Helps in folate
usage and myelin formation. Deficiency: pernicious anemia (lack of intrinsic factor), optic
neuritis.
Ascorbate (vitamin C) reducing agent, hydroxylzes proline and lysine in collagen. Deficiency
(scurvy, breakdown of connective tissue).
Fat Soluble Vitamins
β-carotene (from plants) is broken down in intestines to retinol or Vitamin A. Preformed vitamin
from animal products. Deficiency: night blindness, hyperkeratosis, xerophthalmia.
Vitamin D, not essential if exposed to sunlight. Principal role of vitamin D is in calcium
absorption and coenzyme for parathyroid hormone.
Tocopherols (vitamin E): antioxidant.
Vitamin K is needed in the coagulation cascade. From diet and intestinal bacteria.
39
Regulation of Enzyme Acivity_8
8. Regulation of Enzyme Activity
1) Substrate concentration, i.e. Michaelis-Menton control
2) Competitive inhibition (e.g., looks like substrate) and Irreversible inhibition, (e.g., poisons).
3) Allosteric inhibition (non-competitive) by end-product negative feedback, (e.g., products of
glycolysis, inhibit PFK).
4) Allosteric activation by metabolites e.g. PFK activated by AMP.
5) Association with a repressor subunit or modifier, e.g. G protein complexes.
6) Cooperativity
7) pH, ionic conditions, temperature, e.g. pepsin, calmodulin
8) Reversible (phosphorylation) or irreversible covalent modification, (zymogen proteolysis.
9) Change of enzyme synthesis or degradation rate, genetic control.
Enzyme Inhibition
Types (1) catalytic site, i.e. competitive
(2) allosteric site, i.e. non-competitive
Competitive inhibitors compete directly with the substrate for the catalytic site.
E+I↔ EI
E + S↔ES→ E + P
V vs [S] ± I Reciprocal Plot
If [S]>>[I] Vi=Vmax, so most inhibition at low[S] which would raise Km.
1
x=−
[I ]
Km(1 + )
Ki
Those inhibitors that produce the greatest increase in Km have the best affinity for the ENZ, i.e.
low Ki.
Example: methotrexate competes with folate for dihydrofolate reductase which catalyzes the
formation of thymidine triphosphate for DNA. Used as cancer chemotherapeutic agent.
Sulfa drugs- structural analog of PABA, which is needed by bacteria to make folic acid. Humans
can absorb folate, bacteria cannot.
Noncompetitive inhibitors bind to enzyme or enzyme –substrate complex. Site is separate from
active site (Allosteric). Alters enzyme conformation to inhibit formation of product. Usually
40
Regulation of Enzyme Acivity_8
does not affect substrate binding, i.e. does not affect Km. If it does affect substrate binding it is
called mixed inhibition. Most Non-competitive inhibitors do not resemble the substrate.
E + I⇔ EI
E + S ⇔ES⇔P
ES⇔ESI
Irreversible inhibitors bind covalently, typical of poisons.
Clinical examples:
Ethylene glycol is catalyzed to oxalic acid (severe acidosis) by alcohol dehydrogenase. Give
ethanol. (Competitive inhibition)
Inhibitors of acetylcholinesterase (Ach→choline + acetate). Physostigmine is a competitive
inhibitor used in surgery as a muscle relaxant. Organophosphorus compounds are irreversible
(non-competitive) nerve gases permanently inactive acetylcholinesterase.
Metabolic Pathways
Allosteric regulation (Usually refers to multi-subunit enzymes with sigmoidal kinetics)
1) The reaction catalyzed by the allosteric enzyme is the rate-limiting or committing step
2) Intermediate steps are committed ahead of time, but controlled by #1.
Substrate Positive Cooperativity: binding of one substrate enhances the next. These enzymes
are essentially inactive at low [S], but respond quickly to increases in [S].
Sigmoidal V vs. [S] curve is linearized by use of a “Hill plot” to determine the degree of
cooperativity.
Vi
log = n log[ S ] − log K '
V max − Vi
41
Regulation of Enzyme Acivity_8
Non-substrate modulators can change substrate affinity or degree of cooperativity. Example
AMP binding to PFK raises the affinity of all binding sites to the max., thereby destroying
cooperativity and raising the reaction rate at low [S]. In contrast AMP, converts a simple
Michaelis-Menton enzyme to cooperative, acting as a negative modulator for further purine
synthesis.
PFK activity vs. [S] ±AMP Purine synthesis vs. [S] ±AMP
Regulation by Covalent changes (Chapter 9)
Reversible, e.g. phosphorylation.
Irreversible, e.g. proteolysis
Zymogens
Inactive (proenzyme) that is converted to an active form by specific proteolysis.
Function: storage of inactive form ready for rapid activation when needed, e.g. blood clotting, or
when enzymes need to be transported in their inactive state from site of synthesis to site of
action, e.g. digestive enzymes.
Digestion
1) Endopeptidases: hydrolyze within the chain. Pepsin (gastric); trypsin, chymotrypsin,
elastase (serine proteases secreted by the pancreas). All have different amino acid sequence
specificities.
2) Exopeptidases: Hydrolyze from the ends. Carboxypeptidases hydrolyze from the carboxyl
end (Pancreatic origin). Aminopeptidases are present on the intestinal mucosa.
End result is the production of single, di- and tri- peptides that can be absorbed by the intestinal
mucosa. What’s the mechanism for absorption? Hydrophobic amino acids? Polar amino acids?
a) Pepsinogen (made by gastric glands) is the precursor to pepsin. Inactive at neutral pH.
Weak activity at acid pH. This weak activity starts autolysis of pepsinogen to pepsin and
then pepsin hydrolyzes more pepsinogen at a greater rate.
42
Regulation of Enzyme Acivity_8
b) Pancreatic Proenzymes: Enteropeptidase in intestine cleaves trypsinogen. Trypsin then
activates the other proenzymes.
Pancreas is protected from autocatalysis since 1) proenzymes are made there, 2) packaged in
lipoprotein granules (lipases active in gut), 3) a specific trypsin inhibitor is synthesized by the
pancreas.
Pancreas also secretes HCO3- and lipases. Fats are first emulsified by Bile salts, made in the
liver and stored in the gall bladder. Why need to emulsify fats?
43
Regulation of Enzyme Acivity_8
Blood Clotting
Clotting mechanism must
1) respond rapidly, therefore all precursors are circulating
2) be highly regulated, i.e. not form clots spontaneously.
Platelet adhesion is the initial step in forming a clot causing release of clotting factors (needed
for clotting cascade): ADP, serotonin (vasoconstriction) and eicosanoids (Thromboxanes) from
platelet granules.
Stimuli to adhesion
1) collagen→ ↑intracellular Ca2+→granule release
2) ADP
3) Thrombin
4) Platelet activating factor from WBCs
5) Thromboxane, powerful stimulant to further granule release
Two phases to Clotting
First, form temporary hemostatic plug (just platelets).
Second, conversion to definitve clot is by activation of Factor XII. It is stimulated by exposed
collagen.
Coagulation Pathway (Intrinsic and Extrinsic)
Intrinsic factors are all in the blood. Factor VIII is missing in hemophilia. Cascade of
proteolysis down to Factor Xa. Cascades are irreversible and give considerable amplification.
44
Regulation of Enzyme Acivity_8
Common Pathway: Factor Xa binds to aggregated platelets and cleaves Prothrombin to
Thrombin and Thrombin cleaves Fibrinogen to Fibrin.
Anti-Coagulants (Partial list)
1. Ca2+ required for Prothrombin binding to platelets. Chelators of Ca2+, EDTA or citrate, will
prevent coagulation.
2. Aspirin, inhibits cyclooxygenase in platelets, which makes thromboxane. This retards granule
release and platelet aggregation.
3. Prothrombin uses a postranslationally modified amino acid, γ-carboxyglutamate, to bind Ca2+
and platelet membranes. The carboxylation of glutamate requires vitamin K. Action of vitamin
K is inhibited by dicoumarol and warfarin.
4. Heparin is a complex polysaccharide found in vessel walls that prevent coagulation by
enhancing binding of thrombin to antithrombin. Used clinically.
5. Prostacyclin synthase makes prostacyclins (fish oils) in intima which antagonizes platelet
aggregation. Not present in deeper layers.
Clots dissolved by Plasmin. Dervied from Plasminogen, activated by tissue plasminogen
activator (TPA.).
45
Regulation of Enzyme Acivity_8
REVERSIBLE COVALENT MODIFICATION
Example:
Phosphorylation: serine, threonine, histidine, tyrosine.
(other examples in book)
Protein kinases and phosphatase give exquisite control.
Often phosphorylation starts a cascade: 1) catalytic
amplification, 2) Cascade amplification.
Mutations that cause enzyme to be constituitively
active are out of control (e.g. oncogenes).
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Cell Structure and Function_9 V515
9. Cell Structure and Function
1. Plasma Membrane
a) Phospholipids, cholesterol, glycolipids (e.g. Blood antigens)
b) Proteins and Glycoproteins (e.g. glucose receptor, hormone receptor, Blood Ag, transport
proteins)
c) Function is to maintain internal milieu
2. Endoplasmic Reticulum
a) Smooth ER i) site of fatty acid synthesis
ii) microsomal detoxification enzymes (e.g. liver cytochrome P450)
b) Rough ER: site of protein synthesis. Most abundant in i) secretory cells, e.g. Pancreas
and ii) plasma cells (make Ab).
3. Free ribosomes: mixture of RNA and protein. Involved in protein synthesis of cellular
proteins, e.g. actin, glycolytic enzymes and mitochondrial proteins.
4. Golgi
a)Transport secretory proteins from RER
b)Modify proteins, i.e. post-translational modification (phosphorylation, gylcosylation,
cleave to active form, e.g. insulin).
c) form secretory vesicles for immediate exocytosis
d) fuse with others to form storage vesicles for later release (wait for signal). Example:
insulin
e) contain outer membrane proteins or lysosomal proteins which fuse with each structure.
5. Lysosomes (bud off from golgi)---Garbage Disposal
a) Degradative enzymes: RNAse, DNAse, phosphatases, proteases, lipases, glycases.
b) pH ~5 due to proton pump in membrane. Enzymes work best at this pH.
Tay-Sachs Disease: missing a lysosomal glycolipid hydrolase which leads to enlarged
lyosomes that disrupt cells, mainly in the liver.
c) Endocytosis of extracellular material, fuse with lysosome
e.g. Macrophages (lysozyme degrades peptidoglycan of bacterial cell wall, H2O2, O2-.
Other examples: lipid receptor complexes, viruses.
47
Cell Structure and Function_9 V515
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Cell Structure and Function_9 V515
6. Nucleus
-Chromosomes: DNA-protein complex, gene expression through production of mRNA.
-Nucleolus where rRNA is made.
-Nuclear membrane is continuous with the ER
-DNA replication controlled by cell cycle machinery G1, S, G2, M.
7. Mitochondria
-ATP production machinery
-DNA coding for some mitochondrial enzymes, maternally inherited
-Leber’s optic neuropathy, External Ophthalmoplgia.
8. Amorphous cytoplasm -Scaffold or cytoskeleton contains microfilaments (actin and other
proteins) Microtubules (tubulin), Metabolic enzymes.
CYTOSKELETON / EXTRACELLULAR MATRIX
Cytoskeleton Function: Give structure and shape, control cell movement, movement within cells
& cell division.
Made up of microtubules, microfilaments and intermediate filaments.
Microtubules are hollow cylinders (form highways) by which molecules and cell parts are
actively moved from one site to another, e.g. axoplasmic flow, mitosis.
Made up of α- and β- Tubulin which form protofilaments that aggregate into tubes. Tubes have
polarity (“+” and “-“ ends).
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Cell Structure and Function_9 V515
Microtubule associated proteins (MAPs) Function: stabilize
microtubule, move along it (need ATP) carrying organelles
and molecules, e.g. Motor proteins: cytoplasmid dyneins and
kinesins. Kinesins transport their cargo toward the plus end
and dyneins move toward the minus end. Provide for
movement in opposite directions, e.g. retrograde and
anterograde axon movement. Outer segments of
photoreceptors are dependent on this movement.
Plant alkaloids such as colchicine, vincristine and
vinblastine bind to tubulin molecules and prevent
polymerization. Leads to rapid disappearance of mitotic
spindle. Used in cancer therapy. Taxol stabilizes the
microtubule, thereby arresting the cells in mitosis.
Microfilaments (Actin)
G-actin subunits assemble into f-actin filaments. α-actin in
muscle and β-actin in non-muscle cells. In non-muscle cells
they form the cortex.
Actin filaments and microtubules act together to polarize
some cells. Example killer T-cells. When activated cortex
reorganizes and signal to microtubules to bring Golgi up to
point of contact. Then Golgi release cytotoxic substances.
Actin filaments are responsible for cell movement. Example,
migration following wounding.
Intermediate filaments are fibrous proteins like keratin,
desmin, vimentin, lamin. Tend to concentrate around the
nucleus giving it shape and structure. They are regulated and
will dissolve when phosphorylated in preparation for mitosis.
Intermediate fibers also found in epithelia linking cell to cell.
Help provide mechanical strength.
CELL-CELL Contacts and Extracellular Matrix
Junctions
Gap junction proteins form little holes between cells. Allow for coupling and communication.
Example, lens fibers, corneal epithelium.
Tight junction Complex (many proteins). Attach to actin cytoskeleton. Occludin modulates
permeability around cells. Polarizes epithelial cell into apical and basolateral domains.
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Cell Structure and Function_9 V515
Cell to cell anchors are called desmosomes. Cell to basement membrane junctions: focal
contacts, adhesion plaques, or hemidesmosomes (fibronectin and laminin).
Collagen is the main extracellular structural protein. Made by fibroblasts it forms a triple helix
(very strong). It has postranslationally modified amino acids, hydroxyproline and
hydroxylysine. Hydroxyproline allows for extra stabilizing hydrogen bonds. Hydroxylysine
provides attachments for glycosylation. Proline hydroxylation requires Vitamin C (deficiency is
scurvy).
Glycosaminoglycans (GAGs) are extracellular polysaccharides. All except hyaluroinc acid are
bound to proteins like collagen.
GAGs are highly negatively charged. This attracts osmolytes and water. Gives connective tissue
resiliency. Hydration important for corneal transparency.
GAG production is controlled by thyroid hormone. Deficiency of thyroid hormone causes a
condition called myxedema due to excess GAG formation.
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Membranes_10 V515
10. Membranes
Function: Permeability Barrier
Structure: Bilayer with hydrophobic portions inside and polar groups (Phosphates facing aqueous
environment.
Composition
1) Phospholipids (see Lipids ~page 13)
a) lecithin, e.g. Phosphatidylcholine, -serine, -ethanolamine
b) Inositol phosphates and arachadonic acidfound on inner side, used in signaling
2) Cholesterol- diminishes the lateral mobility of lipids and proteins in the membrane.
3) Proteins
a) surface proteins bound to outer leaflet or attached to special lipids buried in the
membrane. Proteoglycans have large amounts of sugars giving a deep
hydrophilic surface called a glycocalyx. Corneal epithelium.
b) Transmembrane proteins poke into inside and outside spaces. Hydrophobic α-
helices usually span the membrane. A Hydropathy plot shows the average
hydrophobicity of short peptide segments. Used to predict whether stretch is
transmembrane. Outer portion of transmembrane protein has sugars.
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Membranes_10 V515
Functions of transmembrane proteins:
1. Structural links to cytoskeleton and adjacent cells, e.g. Occuldin, a portion of the tight
junction.
2. Transmembrane transport: endocytotic pits, ion channels, carriers, pumps.
3. Receptors for Signal Transduction, i.e., transmit information.
Fluid-Mosaic model of plasma membrane: high lateral mobility, flip-flop frequency low.
Spatial Organization of cell is defined by permeability barriers.
e.g. mitochondria are impermeable to many substrates and products of CAC, Very low
intrinsic permeability to protons.
e.g. Lysosome low pH can only be maintained in a defined space.
Transport: Functions
Nutrients in, waste out
Specialized cell products out
Detoxification
Ion and electrical gradients
Transport: Mechanisms
1. Across but not through membrane: Endocytosis: e.g., phagocytosis and receptor mediated.
a. Phago_ uptake of particulate matter, e.g. bacteria.
b. Receptor mediated- specific molecules are brought in via clathrin-coated pits and specific
surface receptors, e.g. lipoproteins.
Exocytosis is the fusion of intracellular membrane vesicles with plasma membrane, e.g.
secretory vesicles, neurotransmitters.
2. Transport through membrane.
a) Passive Diffusion: flux of a molecule, j (moles/time), across a membrane is proportional to
the driving force, dc/dx, and diffusion coefficient, D. D determined by size, temperature and
solubility (or partition). C is concentration and x is distance.
Fick’s Law of Diffusion: (A is area)
j= DA dc/dx
Speed of diffusion is described by the diffusion coefficient, D. Relation is: X2 = 2Dt. For average
molecule, D= 10-5 cm2/sec,
For x = 1 um, 0.5 ms
100 um, 5 sec
1 mm, 8.3 min
1 cm, 14 hours
Cornea, 500 um, 125 sec.
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Membranes_10 V515
b) Protein mediated transport
1) more rapid than diffusion
2) Saturation kinetics
3) Chemical specificity
4) Related compounds can compete
5) Allosteric inhibition is possible.
b1) Facilitated diffusion
1. no metabolic energy is directly required
2. driven by electrochemical gradients
3. cannot concentrate substance against electrochemical gradient
Examples: facilitated glucose transport, ion channels.
b2) Active Transport
Primary active transport uses ATP directly, e.g. Na+-K+ATPase
Secondary active transport utilizes the electrochemical gradient of some other ion in
order to move against its own electrochemical gradient, e.g. Na+/H+ exchange.
Define: carrier, uniporter (pump), symport, antiport.
Other examples: MDR used to get rid of toxic compounds. Upregulated in cells exposed to
chemotherapeutic agents.
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Signal Transduction_11 V515
11. SIGNAL TRANSDUCTION
Extracellular Signals (from blood or release from neurons)
1. Lipid soluble: Steroid Hormones, e.g., estrogen, testosterone, corticosterone: pass through
membrane and bind a cytoplasmic or nuclear receptor which shuttles to the nucleus and
regulates gene expression.
2. Non-lipid soluble signals: e.g. amino acids, peptides, catecholamines, acetylcholine….bind to
a membrane receptor protein which transmits signal inside. Some generate second
messengers, which triggers signal transduction pathway. They can also change the
concentration of “response element” binding proteins that go to the nucleus and affect gene
expression.
Membrane Receptor Types
1. Ligand gated ion channels, e.g., acetylcholine for Na, Gamma amino butyric acid (GABA)
for Cl-.
2. Receptors with catalytic activity, e.g. the insulin receptor has tyrosine kinase activity.
3. Receptors coupled to production of Cyclic nucleotides, cAMP, cGMP. These change
phosphorylation levels of proteins. Uses a “G” protein (requires GTP) that is located on the
inner leaflet of the memebrane.
4. Receptors coupled to phosphatidylinositol
Acetylcholine (muscarinic receptors)
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Signal Transduction_11 V515
(+) (-)
Gs Gi
AC
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Signal Transduction_11 V515
PKC
PLC
CaM Kinase
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Signal Transduction_11 V515
58
Glycolysis, CAC, OxPhos_12 V515
12. Glycolysis, Citric Acid cycle & Oxidative Phosphorylation
Bioenergetics
How energy is formed from food.
For a reaction to go, there has to be a net reduction in the energy content, i.e. ∆G is negative.
A+B↔C+D
∆G is +, endergonic, gain of free energy,
∆G is - , exergonic, loss of free energy
∆G = 0 at equilibrium
∆G = ∆G0` + RT ln [C][D]/[A][B]
∆G0’ is called the Standard free energy at 37°C. If a reaction is at equilibrium (i.e., formation of
C & D is equal to formation of A & B), then ∆G = 0.
When ∆G = 0, ∆G0 = −RT ln [C][D]/[A][B]. But remember that [C][D]/[A][B] = Keq. So,
∆G0 = -RT lnK. Therefore, ∆G0 is a constant. If K is greater than 1, i.e. [C]x[D]>[A]x[B] at
equilibrium, then ∆G0 will be negative.
∆G tells you whether a reaction can proceed. It does not tell you how fast it will go. Rate is
determined by the reaction-enzyme rate constant Krat and the substrate concentration.
Endergonic processes can proceed when coupled to exergonic, so that the net ∆G is negative.
In biology, energy is typically coupled to high energy phosphates. For example,
∆G0
Phosphoenolpyruvate ↔enolpyruvate + Pi -61.9 KJ/mol
ATP↔ADP + Pi -30.5
Glucose-6-Phosphate ↔glucose + Pi -13.8
Coupling Example: creatine phosphate → creatine + Pi -43.1 KJ/mol
ADP + Pi → ATP +30.5
Net reaction: creatine-Pi + ADP ↔ ATP + creatine net ∆G0’= -12.6
Reverse reaction needed to regenerate creatine-Pi net ∆G0’= +12.6
How does it happen? (need high [ATP]).
These High energy phosphate compounds are called phosphagens.
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Glycolysis, CAC, OxPhos_12 V515
Metabolic Control
1) change the amount of enzyme, slow (10 min to days)
2) Alter kinetics
a) Covalent modification of enzymes, e.g. phosphorylation is rapid. Effected generally by
hormones.
b) Allosteric effectors, metabolites. Provide very rapid precise rate control.
Steps with high negative ∆G0’ will be essentially irreversible. These steps are usually highly
regulated with allosteric and covalent modification used to control. Many exhibit sigmoidal
kinetics, giving more rapid rate changes in the physiological range.
GLYCOLYSIS
Location: Cytosol
Functions:
1) produce energy, i.e. ATP, directly
2) produce pyruvate, which can be oxidized for more ATP in mitochondria
3) produce intermediates needed for biosynthesis, (e.g., glyceraldehyde → glycerol).
Hormone Control (Brief synopsis)
1) Insulin, secreted by β-cells of pancreas in response to rise in plasma glucose. It increases
storage of energy in the form of glycogen, fat and protein.
2) Glucagon, secreted by α-cells of pancreas in response to low glucose. It increases
gluconeogenesis and decreases utilization of glucose.
3) Epinephrine “fight or flight” hormone, mainly affects muscle. Makes energy available, i.e.
increases plasma glucose.
Transport of Glucose
Facilitated diffusion. (Na-coupled used in gut.) Glucose transporter family is called glut. Most
glut transporters (#1-3) are not sensitive to insulin. Glut4, however is insulin sensitive (skeletal
muscle, adipose tissue). Need to keep cytoplasmic [glucose] low to promote glucose uptake.
1) Glucose + ATP → glucose 6-Pi + ADP;
a) Hexokinase: low Km (.001-.1 mM) not sensitive to glucose levels, has constant supply
b) HK inhibited by glucose 6-Pi so won’t deplete plasma glucose supply.
c) Glucokinase found in the liver has simoidal kinetics and high Km (5-10 mM), therefore it
is sensitive to glucose levels in blood. Not inhibited by glucose-6-Pi.
Next step (G 6-P ↔ F 6-P) ∆G0 is close to 0, so the reaction could easily go in either direction.
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Glycolysis, CAC, OxPhos_12 V515
2) Fructose 6-Pi + ATP → Fructose 1,6 Pi +ADP
a) Phosphofructokinase: MAJOR CONTROL SITE. Michaelis-Menten (active) and
sigmoidal kinetics (less active)
b) Inhibited by ATP (at normal cytosolic [ATP]), exacerbated by citrate.
c) Activated by AMP and fructose 2,6-bisphosphate (under hormonal control)
Inhibition at this point backs up fructose 6-Pi, increasing glucose 6-Pi, which turns off HK.
Production of Fructose 2,6-bisphosphate is catalyzed by PFK-2. PFK-2 activity is increased by
insulin in the liver and by epinephrine in muscle. Epinephrine increases cAMP in muscle cells.
This causes phosphorylation of PFK-2 increasing its activity, giving more fruc 2,6-bisphosphate
and thereby activating PFK-1.
3) Intermediate steps are at equilibrium. (For all the steps, see your book)
Aldolase catalyzes fructose 1,6 bisphosphate to 2 trioses, glyceraldehyde 3-Pi and
dihydroxyacetone-Pi, which is a precursor for α-glycerol Pi for lipid synthesis.
2 trioses + 2NAD+ → 2 trioses + 2NADH
2 trioses + 2 ADP → 2 3-PHOSPHOGLYCERATE + 2 ATP →→
4) 2Phosphoenolpyruvate + 2ADP → 2Pyruvate + 2ATP ∆G0’ = -31.4 KJ/mol
a) This step is catalyzed by Pyruvate Kinase activity is not controlled much except in the liver
where a different isozyme is under strict control, which is important for gluconeogenesis.
Balance sheet: get 2 NADH, 4ATP, use 2 ATP
net get 2 NADH, 2 ATP.
Pyruvate can then enter mitochondria for further metabolism that requires oxygen or if oxygen is
unavailable or cells lack mitochondria, then:
2Pyruvate + 2NADH ↔ 2lactate +2 NAD+
a) This is catalyzed by Lactate Dehydrogenase (LDH), the reaction helps regenerate NAD+ for
use in the intermediate steps of glycolysis.
Thus Anaerobic glycolysis net reaction is: glucose + 2ADP + 2Pi → 2Lactate + 2ATP
Some tissues always produce lactate: RBCs, cornea, lens.
ATP hydrolysis yields protons (i.e. acid). However ATP production in mitochondria consumes
protons, thus no net acid is produced.
ATP + H2O ↔ ADP + Pi + 2H+
However, during hypoxia (lack of oxygen or if there are no mitochondria) protons produced
during hydrolysis are not subsequently consumed. This can lead to metabolic acidosis and since
it is linked to lactate production it is called lactic acidosis.
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Glycolysis, CAC, OxPhos_12 V515
If oxygen is available, pyruvate + NAD+ + CoA → Acetyl CoA + NADH + CO2
This step occurs in the mitochondria. CoA is derived from the water soluble vitamin
pantothenic acid.
a) This step is catalyzed by pyruvate dehydrogenase (PD). PD requires the coenzyme
thiamin.
b) PD is inhibited by Acetyl CoA and NADH, which can come from other sources, e.g.
lipid breakdown.
c) PD is inhibited by phosphorylation via pyruvate dehydrogenase kinase. This kinase
is inhibited by pyruvate and stimulated by Acetyl CoA.
Clinical Note:
Alcoholics are thiamin deprived → Lactic acidosis.
Alcoholics can also present with Nutritional Amblyopia
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Glycolysis, CAC, OxPhos_12 V515
Regulated
only in
liver
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Glycolysis, CAC, OxPhos_12 V515
CITRIC ACID CYCLE (located in mitochondria)
Function: Produce reducing equivalents: NADH, FADH and GTP
First Step: Acetyl CoA + oxaloacetate → Citrate
Acetate for acetyl CoA can come from many substrates.
Major Regulatory Step is:
Isocitrate + NAD+ → α-ketoglutarate+ NADH
a) Isocitrate Dehydrogenase is inhibited by NADH, shifts sigmoid curve right.
b) ADP stimulates, shifts curve left.
c) Inhibition at this step causes accumulation of citrate, which diffuses back to cytosol
and inhibits PFK
Therefore, the CAC is regulated primarily by [NADH] and [ADP]
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Glycolysis, CAC, OxPhos_12 V515
OXIDATIVE PHOSPHORYLATION
Function: take reduced equivalents (NADH, and FADH) and produce usable energy in the form
of ATP.
F0
channel
FADH
There are three major steps with sufficient energy drop to produce ATP from ADP.
1) NADH to coenzyme Q
2) Cytochrome b to c
3) Cytochrome a/a3 (oxidase, Complex IV) to O2
NADH dehydrogenase is reduced by NADH. Reduced NADH dehydrogenase then passes the
electrons to Coenzyme Q, thus reducing it. Reduced Coenzyme Q passes electrons to the b-c1
complex. Reduced cytochrome c then passes electrons to cytochrome oxidase, which contains
the binding site for oxygen. Oxygen accepts 4 electrons as it is reduced to water.
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Glycolysis, CAC, OxPhos_12 V515
Cytochromes contain Heme and electrons are transferred to Fe3+ to make Fe2+.
The poisons antimycin, rotenone & cyanide work on cytochromes.
The energy released from the electron transfer is used to shuttle hydrogen ions out of the inner
matrix against its gradient. Therefore, pH is alkaline in mitochondria relative to cytosol. This
also creates a negative membrane potential.
Protons come back through the F0 channel, activate F1 ATP synthase
H+ + ADP + Pi → ATP
*A maximum of one ATP can be produced from the proton pumping at each of the three
membrane-spanning complexes, i.e. 3ATPs made from each NADH, 2ATPs made from each
FADH.
Rates controlled by [ADP]. Low ADP with plenty of other substrate gives low oxygen uptake.
Adding ADP speeds up oxygen consumption.
ATP is translocated out in exchange with ADP.
Dinitrophenol (DNP) is a mitochondrial poison. It acts as a proton pore. Protns shuttled out by
the membrane complexes will simply go back in if DNP is present. Thus DNP effectively
uncouples H+ translocation from ATP synthesis.
Overall Balance: glucose + 6O2 + 36Pi + 36 ADP → 6CO2 + 36 ATP + 42 H2O
Glucose + 6O2 → 6CO2 + 6 H2O ∆G0’ = -680 Kcal
36Pi + 36ADP → 36ATP + 36H2O ∆G0’ = +263 Kcal
So Efficiency = 263/680 = 39%.
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Glycogen Metabolism_13 V515
13. Glycogen Metabolism
Glycogen is the storage form of glucose. All cells have some, but is most concentrated in liver
(up to 8%) and muscle (up to 2%).
Why make polymer? (reduce osmotic effect).
1) Glucose 6-Pi ↔ Glucose 1-Pi
2) Glucose 1-Pi + UTP ↔ UDP-Glucose + PPi; PPi → 2Pi
3) nGlycogen + UDP-Glucose → n+1Glycogen + UDP; Glycogen synthase
Links are α-1,4. A branching enzyme adds α-1,6 links every 4 glucose residues.
Glycogen breakdown uses other reactions
1) Phosphorylysis at α-1,4 link producing glucose 1-Pi is catalyzed by phosphorylase.
2) glucose 1-Pi is converted to Glucose 6-Pi by phosphoglucomutase. Glucose 6-Pi can go
to glycolysis (muscle) or be converted to glucose by glucose 6-Phosphatase for plasma
glucose (liver).
Von Gierke’s disease (Type I Glycogen Storage Disease) missing glucose 6-Phosphatase;
accumulate Glucose 6-Pi. Goes through glycolysis → excess lactate or goes back to glycogen
synthesis and get enlarged liver. Result is hypoglycemia.
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Glycogen Metabolism_13 V515
Control of Glycogen Synthesis
1) covalent : Phosphorylated Glycogen synthase is inactive. True for liver & muscle.
2) Allosteric: Glycogen synthase is activated by high levels of glucose 6-Pi (possibly only in
liver).
3) Glycogen synthase & Phosphorylase are associated with glycogen. As glycogen gets larger
more glycogen synthase and phosphorylase are phosphorylated, thereby limiting glycogen
build up. In muscle, limited to 2% wet weight, liver 8%.
Control of Glycogen Degradation
1) Phosphorylase is activated by phosphorylation.
2) Dephosphorylated Phosphorylase is activated by AMP in muscle only. Why? (isozyme in
liver is not affected by AMP). Liver doesn’t need to respond to cell energy status.
Hormonal effects on Phosphorylation
Epinephrine (fight or flight) increases cAMP in muscle (β-receptor), which activates protein
kinase A (PKA).
a) PKA phosphorylates glycogen synthase, inactivating it. Result: slows glycogen
synthesis.
b) PKA phosphorylates Phosphorylase Kinase (in) giving Phosphorylase Kinase (ac).
Phosphorylase kinase phosphorylates phosphorylase making it active and breaking
down glycogen. (this represents a small cascade effect).
c) Muscle contraction (increased calcium) can activate phosphorylase kinase
allosterically. This provides local control where needed.
In liver epinephrine activates an α-adrenergic receptor. This is coupled to a G-protein that
activates Phospholipase C causing production of Diacylglycerol and IP3. DAG causes increased
Protein kinase C activity and IP3 raises Ca2+ leading to activation of calmodulin dependent
protein kinase, both of which act to phosphorylate glycogen synthase. It also will phosphorylate
phosphorylase kinase. Thus glycogen synthesis is inhibited and glycogen breakdown is
activated.
Glucagon binds to its own receptor in liver, increasing cAMP.
Insulin lowers cAMP levels (maybe by activating phosphodiesterase), so it will encourage
glycogen synthesis and discourage glycogen breakdown.
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Glycogen Metabolism_13 V515
(Muscle)
Liver
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Glycogen Metabolism_13 V515
PENTOSE PHOSPHATE SHUNT
Function: 1) provide NADPH for biosynthesis (lipids) and reducing equivalents for free radical
scavengers. 2) produce ribose 5-phosphate for nucleotide and coenzyme synthesis.
In the Eye, Free radical scavenging by glutathione in the crystalline lens is very important.
Glutathione is a tripeptide with a cysteine group.
2G-SH + H2O2 → G-S-S-G + H2O (catalyzed by Glutathione Peroxidase)
Regenerate reduced glutathione: G-S-S-G + NADPH → 2G-SH + NADP+ (catalyzed by
Glutathione Reductase)
Other Hexoses
Fructose (fruit, sucrose), galactose (from lactose).
Both are taken up by the liver and converted eventually to glucose or glycogen.
Galactose is converted to Glucose 6-Phosphate. Enzymes for this process are not expressed
outside the liver.
Fructose is converted to glycolytic intermediates Glyceraldehyde-3-Pi and dihydroxyacetone –Pi
or up to Glu-6-Pi and released as glucose or stored as glycogen. Again, enzymes for this process
are liver specific.
Galactosemia (enzyme gal-1-Pi uridylyltransferase is missing, see book for details if interested.)
Liver accumulates gal-1-Pi.
Excess galactose in the eye (lens) is converted to galactitol by adlose reductase.
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Gluconeogenesis_14 V515
14. GLUCONEOGENESIS
Purpose: Provide plasma glucose from lactate, pyruvate, amino acids, glycerol, fructose and
galactose.
Physiological Function: keep [glucose] up during fasting.
Location of pathway: liver and kidney cortex.
4
3
2
1
Four unique enzymes are required, the rest are simple reversals of glycolytic steps. The unique
reactions are at points where the glycolytic steps are essentially irreversible, i.e. high –∆G.
So how to get phosphoenol pyruvate from pyruvate? The glycolytic reaction (shown below) is
hard to reverse:
PEP + ADP ⎯⎯→ pyruvate + ATP
PK
∆G0= -7.4kcal
Answer: Use two steps with ∆G0~0 to get around it, but need to put energy into the process.
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Gluconeogenesis_14 V515
Gluconeogenic Step 1) Pyruvate + ATP + CO2 ⎯⎯ ⎯ ⎯ ⎯ ⎯ ⎯→
PyCarboxylase , AcCoA
oxaloacetate + ADP
∆G0~0
Gluconeogenic Step 2) Oxaloacetate +GTP ⎯
⎯⎯⎯⎯⎯ → PEP + GDP + CO2
kinase
PEPcarboxy
∆G0~0
PEP is then converted to Fructose 1,6-Biphosphate by simple reversal of glycolytic steps.
PEP→→→F-1,6-BP
How to get F 6-P from F 1,6-BP?Below is the glycolytic step:
ATP + F-6-P ⎯PFK → ADP + F-1,6-BP
⎯
⎯ ∆G0= -3.4kcal
F −1, 6 − Bisphosphatase
Gluconeogenic Step: F-1,6-BP + H2O ⎯⎯⎯⎯⎯⎯ → F-6-P ⎯ ∆G0= -4.0kcal
F-6-P↔G-6-P
How to get glucose from glucose 6-P?Below is the glycolytic step:
Glucose + ATP ⎯⎯⎯ → G-6-P + ADP
⎯
glucokinase
∆G0= -4.0kcal
G − 6 − Phosphatase
Gluconeogenic Step: G-6-P ⎯⎯⎯⎯⎯ → Glucose + Pi
⎯ ∆G0= -3.3kcal
Energy is required for gluconeogenesis. 6 high energy phosphate bonds used to convert 2
lactates to 1 glucose.
OTHER SOURCES FOR GLUCOSE
1) Glycerol, biproduct of triacylglycerol breakdown, can be converted to glycerol phosphate and
then oxidized to DHAP, thereby used to generate glucose.
2) Amino acids that can breakdown to glycolytic intermediate, e.g. alanine, or a net citric acid
cycle intermediate are potentially glucogenic.
3) But: No gluconeogenesis from Acetyl CoA
Thus fatty acids cannot make glucose. Adding Acetyl CoA only makes TCA cycle go round,
forming oxaloacetate, which reacts with Acetyl CoA again.
Control of Gluconeogenesis
1) Pyruvate carboxylase is stimulated by Acetyl CoA. Acetyl CoA also inhibits pyruvate
dehydrogenase. Thus Acetyl CoA stimulates gluconeogenesis and inhibits metabolism of
pyruvate to citrate. High [Acetyl CoA] is a consequence of fatty acid metabolism that
accompanies fasting. So indirectly, fatty acid breakdown stimulates gluconeogenesis.
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Gluconeogenesis_14 V515
2) PEP Carboxykinase activity is controlled by its synthetic rate, i.e. no allosteric or covalent
control. Transcription of the gene for PEP Carboxykinase is increased by increases in cAMP
levels (i.e. phosphorylation of stimulatory transcription factors). [cAMP] is increased by
Glucagon in the liver.
3) Liver pyruvate Kinase has additional amino acids that give it both allosteric and covalent
control. It also has sigmoidal kinetics. (Figure 13.4 in book)
Effectors of Liver pyruvate kinase:
a) F-1,6,BP This indicates glucose inflow, so it activates PK.
b) Alanine indicates protein catabolism. This is negative for PK.
c) Phosphorylation of PK is a negative regulator. Phosphorylation is via Protein Kinase A. Thus
the increased [cAMP] due to glucagon or epinephrine will slow glycolysis and thus favor
gluconeogenesis.
4) PFK inhibited by ATP, citrate, activated by F-2,6-bisphosphate
PFK − 2
F-6-P ⎯⎯⎯→ F-2,6-BP
5) F-1,6-Bisphosphatase in liver is inherently more active than PFK and it is inhibited by F-2,6-
bisphosphate.
If glucose influx to the liver is low (e.g., hypoglycemia) then very little F-6-P will be around, so
there will be very little F-2,6-BP.
6) PFK-2 is active when non-phosphorylated, i.e. insulin is present. It is inhibited (actually
becomes a phosphatase) when phosphorylated, i.e. high [cAMP] from glucagon stimulation.
7) Glucose-6-Phosphatase is inhibited by high glucose, i.e. causes accumulation of G-6-P and
thus glycogen accumulation.
8) Glucokinase has a relatively high Km.
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Fatty Acid Catabolism_16 V515
15. FATTY ACID CATABOLISM
Triacylglycerols (Triglycerides) = Fatty acid + glycerol.
Storage Triacylglycerols are 16 (Palmitic) or 18 (Stearic) carbons.
TAGs arise from the diet or are synthesized in the liver. They are repackaged as lipoproteins
and transported to adipose tissue for storage.
Other lipids include: steroids, phospholipids, glycolipids, sphingolipids, and Viamins A,D,E,K.
Lipid represents the major source of potential energy in the body.
Fatty acids in the body arise from the diet or are synthesized by breaking down TAGs.
There is continual synthesis and breakdown of TAGs depending on the frequency of feeding and
energy demand.
Lipolysis (Adipose cells)
Breakdown of TAGs is controlled by Glucagon, Epinephrine and insulin. Glucagon and
epinephrine increase cAMP, which leads to phosphorylation and activation of hormone sensitive
lipase. Insulin decreases [cAMP].
TAG ⎯hormone − sensitiveLipase→ DAG + FA ⎯⎯⎯⎯→ FFA (unesterified) + glycerol
⎯⎯⎯⎯⎯ ⎯ otherLipases
FFA (free fatty acids) and glycerol leave the adipocyte. FFAs are transported bound to albumin
and go to the liver and other tissues (except brain and RBCs) and are broken down for energy.
Glycerol goes to the liver and is used to make TAGs or glucose.
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Fatty Acid Catabolism_16 V515
FA β-Oxidation, one cycle (net reaction):
FA + FAD + NAD + ATP + CoA → Acetyl CoA + NADH + FADH2
Oxidation occurs in the inner portion of mitochondria, so a shuttle system is needed to move acyl
CoA inside. Figure below illustrates the carnitine shuttle:
Once inside, the Fatty acyl CoA undergoes β-oxidation (β-carbon is oxidized) to acetyl CoA and
reduced coenzymes.
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Fatty Acid Catabolism_16 V515
Net energy Production:
So a 16 carbon FA gives 7 FADH2 and 7 NADH yielding (14 + 21) = 35 ATP; plus 8 Acetyl
CoAs (12 x 8= 96 ATP). Only 1 ATP was used.
Lipids have higher caloric density than carbohydrates and don’t absorb water.
Use of lipids spares glucose by producing lots of acetyl CoA, (which shuts down glucose
utilization).
KETONE BODIES
Acetoacetate and β-hydroxybutyrate (two weak acids) (Figure 14.2)
Plasma concentration 1- 5 mM for ketone bodies and ~ 1mM for fatty acids.
These compounds are formed only in the liver, but can be metabolized in all tissues with
mitochondria (except liver).
They are formed when the transport of Fatty acids into mitochondria is saturated.
This condition can occur during fasting, high fat-low carbohydrate diet, a diabetic crisis or
during starvation.
Net Reaction:
3Acetyl CoA → Acetoacetate ↔ β-hydroxybutyrate + 2 free CoA + Acetyl CoA
The free CoAs help to keep β-oxidation continuing.
Fate of ketone Bodies:
Acetoacetate + NADH ↔ β-hydoxybutyrate + NAD+
β-hydoxybutyrate is more stable. The ketone bodies then leave the liver and enter the blood.
Acetoacetate spontaneously breaks down to CO2 + acetone. Acetone is volatile and leaves via
the lungs.
Use of Ketone Bodies in other tissues:
β-hydoxybutyrate + NAD ↔ Acetoacetate +NADH
Acetoacetate + succinyl CoA (a CAC intermediate) → succinate + acetoacetyl CoA
Acetoacetyl CoA + CoA → 2 acetyl CoA
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Fatty Acid Catabolism_16 V515
Even brain can use ketone bodies (Only RBCs cannot)
Priority for use: ketone bodies> fatty acids> carbohydrates
Excess ketone body production (diabetes) can lead to:
1) lose in urine, i.e. lose energy
2) since negatively charged also lose cations, Na+, K+ and NH4+ (causes dehydration→ polyuria,
polydypsia)
3) acidosis;
a) ketone bodies give up a proton,
b) CO2 can form from acetoacetate
c) dehydration from salt loss also contributes to acid concentration.
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Fatty Acid Synthesis_16 V515
16. LIPID SYNTHESIS
Fatty acids are synthesized from Acetyl CoA under conditions of energy surplus. This occurs
mainly in the liver and to a small extent in adipose tissue.
Since glycolysis is not extensive in the liver, much of the substrate comes from pyruvate and
lactate derived from other tissues, however FA synthesis would be stimulated by a high
carbohydrate diet.
Net reaction:
Acetyl-CoA + NADPH + ATP + biotin +HCO3- → Palmitate (16 carbons)
⎯⎯⎯⎯⎯ ⎯
Pyruvate ⎯pyruvatedehydrogenase→ AcetylCoA
Acetyl CoA (2 carbon) +ATP +CO2 ⎯⎯⎯⎯⎯⎯⎯ ⎯→ MalonylCoA (3 carbon) + ADP + Pi
AcetylCoACarboxylase ,biotin
MalonylCoA + Acetyl CoA + NADPH ⎯⎯⎯⎯⎯→ Palmitate + NADP+ + CO2↑
fattyacidsynthase
FATTY ACID SYNTHASE (FAS) is a multifunctional enzyme having sites for many
intermediate steps of palmitate synthesis in one place.
This takes place in the cytosol/ER. However AcetylCoA comes from the mitochondria and
can’t cross the membrane.
Citrate accumulates in times of sufficient or excess energy in the liver. Citrate can exit
mitochondria via a specific citrate translocase.
Citrate + ATP + CoA ⎯⎯⎯⎯→ oxaloacetate + AcetylCoA + ADP + Pi
citrateLyase
Control of Fatty Acid Synthesis
1) Substrate and product control (pyruvate, citrate, FA)
Why doesn’t citrate formed in mitochondria go into the CAC?
Under conditions of low [FA] (so [AcetylCoA] is low), high carbohydrate diet and insulin,
pyruvate dehydrogenase is active, i.e. lots of Acetyl CoA can be made. So citrate is rapidly
made, CAC cycles and lots of NADH and ATP (low ADP) are made. Elevated NADH and low
ADP will then inhibit isocitrate dehydrogenase causing accumulation of citrate, which is
shuttled to the cytosol.
Citrate is converted to AcetylCoA and oxaloacetate. [Citrate lyase] is higher in high
carbohydrate diets.
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Fatty Acid Synthesis_16 V515
Oxaloacetate + NADH ⎯⎯⎯⎯⎯⎯→ Malate + NAD+
⎯
MalateDehydrogenase
Malate + NADP+ ⎯⎯⎯⎯→ pyruvate + CO2 + NADPH
⎯
MalicEnzyme
Net effect is to transfer reducing equivalents from NADH (high concentrations) to NADP
(needed for fatty acid synthesis).
2) Control at AcetylCoA Carboxylase (converts AcetylCoA to MalonylCoA).
a) citrate stimulates polymerization of the enzyme which is a more active form. (+)
b) Phophorylation of enzyme favors monomers, i.e glucagon (-)
c) Fatty-acylCoA inhibits AcetylCoA carboxylase (-)
Glucagon favors gluconeogenesis, so pyruvate is used to make glucose, not FA.
Fatty-acyl CoA would be coming from exogenous sources, so it is a signal for no need to make
more fatty acids.
Citrate does three things for FA synthesis:
1) it is a substrate for MalonylCoA
2) it is an allosteric activator
3) it is a substrate for malate that gives NADPH that is needed for FA synthesis.
FAS is slow, allowing MalonylCoA to accumulate. MalonylCoA is an inhibitor of carnitine
transferase 1, which is used to breakdown FA. So futile cycle does not occur and FA can form
into TAGs or phospholipids
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Fatty Acid Synthesis_16 V515
Fatty acids can be esterified with α-glycerol phosphate
to form phosphatidic acid.
Phosphatidic acid is the substrate for triacylglycerols
(TAGs) or phospholipids.
Choline can react with phosphatidic acid to give
phosphatidylcholine
Ethanol ingestion leads to fatty liver
Ethanol + NAD+ ⎯⎯⎯⎯⎯⎯ ⎯→ acetaldehyde +
AlcoholDehydrogenase
NADH
This increases the NADH/NAD ratio leading to increased
α-glycerol phosphate and thus TAGs.
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Fatty Acid Synthesis_16 V515
Lipoprotein and Transport of Lipids
TAGs, cholesterol, cholesterol esters and phospholipids have poor solubility in aqueous
solutions. So they are transported in special micelle structures that contain signal proteins.
Chylomicrons are formed in the intestinal epithelium from absorbed fats. They are secreted into
the lymph and dumped into the vena cava. Pizza meal gives milky blood due to their large size.
Chylomicrons are “processed” by lipoprotein lipase on the surface of vascular endothelium of
capillaries in adipose tissue. The released fatty acids are absorbed, glycerol goes to the liver.
This process produces chylomicron remnants, which are taken up by the liver and reprocessed.
Transport of endogenous cholesterol and TAGs is by liver derived lipoproteins.
VLDLs (very low density lipoproteins) are made in the liver. They contain less lipid and a little
more protein than chylomicrons. VLDLs are hydrolyzed by Lipoprotein lipase, particularly at
adipose for storage. As the VLDLs lose TAGs their protein and cholesterol content go up and
their density goes up so they become IDL and then LDL, which is cholesterol rich.
LDL delivers cholesterol to peripheral tissues, especially adrenals, testes, ovaries to make
steroid hormones. The remainder goes to liver for repacking in VLDLs and bile acids or is
deposited on oxidized endothelium by macrophages (foam cells) and cause atherosclerosis.
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Fatty Acid Synthesis_16 V515
HDL is made separately. Highest % of protein , highest density.
HDLs:
a) transfer apolipoproteins to other particles
b) takes up free cholesterol from the surface of cell membranes
c) have an enzyme to create cholesterol ester (less polar, moves to the center) thus removed
from circulation.
d) Taken up by liver where cholesterol is repacked, converted to bile salts or secreted in the
bile.
Ratio of HDL/LDL is used as clinical indicator for atherosclerosis.
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Fatty Acid Synthesis_16 V515
Cholesterol is a precursor of steroid hormones, bile acids and a component of membranes.
Cholesterol is synthesized in the liver predominantly. Liver can synthesize much more than is
ingested.
Cholesterol is synthesized from AcetylCoA.
HMG − CoA − Re ductase
⎯
Acetyl CoA →→→ HMG-CoA ⎯⎯ ⎯ ⎯ ⎯ ⎯ → Mevalonate→→→Cholesterol
Rate limiting step is at hydroxymethylglutaryl CoA (HMGCoA) reductase, which is inhibited by
cholesterol itself or phosphorylation via cAMP. So glucagon inhibits cholesterol synthesis and
insulin stimulates it.
Cholesterol also causes transcriptional inhibition for HMGCoA reductase.
HMGCoA Reductase inhibitors, e.g., Lovastatin, is used to treat hypercholesterolemia.
Greatest loss of cholesterol is via the bile.
1) increase conversion to bile salts
2) slow reabsorption. Cholestyramine is a positively charged resin that binds the negatively
charged bile acids.
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Fatty Acid Synthesis_16 V515
Eicosanoids
Potent hormones with a variety of physiological effects, especially apparent in inflammatory
reactions.
Arachidonic acid is a 20 carbon polyunsaturated fatty acid made from linoleate, which is an
essential polyunsaturated fat from plant oils. Arachidonic acid is incorporated into membrane
phospholipids. Precursor to leukotrienes (Lipoxygenase), thromboxanes and prostaglandins
(Cyclooxygenase, COX). Glucocoticoids (Cortisol, prednisolone) inhibit Phospholipase A,
thereby preventing arachidonic acid formation. Thus they are potent anti-inflammatory drugs.
Leukotrienes, cause WBC aggregation, bronchoconstriction, increased vascular permeability and
T-cell proliferation.
Prostaglandin formation is via two isozymes: Cox-1, constitutive (e.g. on GI epithelium,
platelets)
Cox-2 associated with inflammatory cells.
Thromboxanes stimulate platelet aggregation, vasoconstriction.
PGI, PGE and PGD cause vasodilation, decrease platelet aggregation and increase lymphocyte
migration.
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17. Review of Hormone Action
There is a small set of hormones, but a large variety of cellular and physiological responses.
This variation is due to the variety of receptor subtypes that may be present on each cell.
G Protein coupled receptors.
The major membrane transducer is a G protein (for GTPase). It has three subunits , α, β, and γ.
Example is true for β-adrenergic receptors, glucagon receptor and other polypeptide receptors.
1) Hormone binds the receptor
2) Activated receptor will bind the G protein.
3) This causes GDP associated with the α-subunit to dissociate.
4) GTP takes GDP’s place and the α-subunit dissociates from the G protein and binds to the
internal effector, e.g. adenyl cyclase, which makes lots of cAMP.
5) The α-subunit GTPase hydrolyzes GTP to GDP and the cycle can restart.
cAMP activates catalytic subunit of A-Kinase (also called Protein Kinase A). cAMP is degraded
by Phosphodiesterase. A-Kinase will phosphorylate serine and threonine residues leading to a
change in the activity of the target protein.
Some receptors (α-2 adrenergic) are associated with an inhibitory G protein (Gi), which then
can inhibit adenyl cyclase.
The α-1 adrenergic receptor (and others) are associated with G proteins that activate the
Inositol phospholipid signaling pathway.
1) Hormone binds the receptor
2) Activated receptor will bind the Gq protein.
3) Activated G protein activates Phospholipase C
4) Phospholipase C cleaves PIP2 into IP3 and DAG
IP3 receptors on ER will release Ca2+ on binding. Ca2+ binds to calmodulin and other calcium
binding proteins that can then activate specific enzymes.
DAG activates Protein kinase C, which is a known stimulator of cell proliferation.
Insulin receptor system is independent of G proteins.
Activated insulin receptor on the membrane acts as a tyrosine kinase. Thought to act by
phosphorylating and stimulating protein phosphatases.
1) It’s action is counter to cAMP.
2) Insulin increases the number of glucose transporters (muscle, adipose, liver)
3) Increases the rate of glucose uptake as well.
Steroid receptors are in the cell (cytoplasm or nucleus). [Possible exception is Aldosterone]
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Regulation of Metabolic and Catabolic Pathways
Glucagon
Secreted by the α-cell of the pancreas in response to low plasma [glucose]. The α-cell has a
“glucose sensor”. It requires insulin for glucose to get in. So in diabetics, the α-cell is always
seeing low glucose and thus is secreting glucagon.
When GLUCAGON binds to its receptor it causes an increase in cAMP. Glucagon receptors
are found in liver and adipose tissue.
Adipose: increased cAMP activates hormone-sensitive lipase. = Release of fatty acids.
In Liver increased cAMP leads to:
1) phosphorylation of glycogen synthase and phosphorylase kinase (phosphorylation of
phosphorylase) causing decreased glycogen formation and increased glycogenolysis.
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2) Phosphorylate PFK-2(L), which turns it into a F-2,6-bisphosphatse, lowering F-2,6-
bisphosphate, which is an activator of PFK , thus slows glycolysis and favors gluconeogenesis.
3) By phosphorylation, it inhibits pyruvate kinase (PEP →pyruvate), thus enhancing
gluconeogenesis.
Indirect effects on liver:
1) High plasma FA levels (release from glucagon stimulated adipose cells) cause elevated
Acetyl CoA inhibiting entry of pyruvate into CAC, thus favoring gluconeogenesis.
2) High Acetyl CoA causes formation of ketone bodies. Used in many tissues, so it is glucose
sparing. For example, Fatty acids and ketone bodies used in muscle (which is not sensitive to
glucagon), spares the use of plasma glucose.
3) High AcetylCoA activates Pyruvate Dehydrogenase Kinase which phosphorylates PD (-)
inactivates it..
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EPINEPHRINE
Released by the adrenal medulla in response to stress (sympathetic stimulation). Binds to alpha
and beta-adrenergic receptors.
Skeletal Muscle and Heart (fig 16.9 in book)
1) α-1 receptors cause increased [Ca2+] leading to greater force of contraction.
2) Ca2+ activates phosphorylase kinase leading to glycogen breakdown, thereby giving glucose
for glycolysis, etc.
3) β-receptor stimulation increases cAMP, which phosphorylates phosphorylase kinase leading
to glycogen breakdown.
4) cAMP also leads to Phosphorylation of PFK-2(M), but unlike liver this activates it to form
more F-2,6-bisphosphate, which activates PFK-1.
Thus muscle is not a source of glucose, however glucose is formed and used by the muscle when
needed.
Liver (Fig. 16.10 in book)
1) α-1 receptors cause increased [Ca2+] activates phosphorylase kinase leading to glycogen
breakdown giving glucose, which is dumped into the plasma.
Adipose
1) β-receptors cause increased cAMP which activates hormone-sensitive lipase and release of
Fatty acids. If mental stress, not using energy may contribute to fatty plaque build-up.
β-Cells of the Pancreas
1) α-2 receptor decreases cAMP. cAMP favors insulin release, so epi will inhibit insulin
release.
INSULIN
Secreted in response to high glucose by β-cells of the pancreas. Uptake of glucose, amino acids,
fatty acids, etc. after a meal cause an increase in ATP and NADPH levels in the β−cell. These
molecules will then cause a K+ channel to close, thereby depolarizing the membrane potential.
This activates a voltage sensitive Ca2+channel causing Ca2+ influx. High cytosolic Ca2+ together
with IP3 stimulated production from gut hormone stimulation (CCK and acetylcholine) cause
release of stored vesicles that contain insulin. Insulin release is inhibited by epinephrine
(decreases cAMP through α-receptor), but stimulated by glucagon (increase cAMP through β-
receptors).
Insulin sensitive tissues: muscle, adipose, liver.
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Muscle: insulin will stop glycogen breakdown, reform stores
Adipose:
1) inactivate hormove-sensitive lipase
2) increase in synthesis and secretion to the blood vessel wall of lipoprotein lipase, which
breaksdown TAGs from VLDLs and chylomicrons. Take up fatty acids.
3) Form α-glycerol phosphate for TAG formation
Liver:
1) stop glycogen breakdown, reform it.
2) Slow gluconeogenesis
3) Slow fatty acid breakdown, reform them
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Amino Acid Metabolism_18
18. Amino Acid Metabolism : Urea formation Chapter 17 in book
Nitrogen balance: Cannot be stored, so in adults what goes in must come out. During growth,
however there is net intake of nitrogen. This contrasts with sugars and lipids.
Excess nitrogen is excreted by dying skin, in the feces, and mostly in the urine as Urea and
Ammonium, (NH4+).
Ammonia is not preferred since it is toxic and highly lipid permeable. In contrast, urea is non-
toxic and about 40% is excreted with each pass through the kidney. During acidosis, more
ammonia is in the form of ammonium, NH4+, which is not permeable.
Urea is made in the liver. The precursor is NH4+ and aspartate.
Sources of NH3
In the mitochondria:
1) Glutamate + NADP + ←⎯ ⎯ ⎯ ⎯ ⎯⎯→ α − ketoglutar ate + NADPH + NH 3
glutamate dehydrogenase
This reaction has +∆G, but α-ketoglutarate is metabolized (glucogenic) and ammonia is
converted to urea.
⎯⎯⎯
2) Gluta min e ⎯gluta min ase→ glutamate + NH 3 (mitochondria)
This occurs in the liver (form urea) and the kidney (form NH4+).
3) The other amino acids transfer their nitrogens via transamination reactions that make
glutamate. Glutamate can then either form ammonia or enter the urea cycle.
Example: α-ketoglutarate + aspartate ↔ glutamic acid + oxaloacetate
Reactions 1 and 3 are reversible, so they can be used to breakdown or form amino acids.
Urea Cycle (in mitochondria) (see fig 17.2 in book)
Ammonia + CO2 + ATP →→→→urea
The last step is hydrolytic cleaving of arginine by arginase to urea and ornithine. Urea goes to
the kidney for excretion and the orinithine re-enters the mitochondria to restart the urea cycle.
1) Regulation of arginine breakdown: Arginase has a high Km for arginine so it is broken down
only when in excess in the liver.
2) All the enzymes of the urea cycle are adaptive. Thus they increase their amounts in response
to high protein diets, cortisol treatment and diabetes.
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Amino Acid Metabolism_18
METABOLISM OF SELECTED AMINO ACIDS
Glutamine (can be made from glutamate)
Glutamate + NH3 + ATP → Glutamine +ADP, catalyzed by glutamine synthetase, thus it can
detoxify ammonia.
Also, glutamine synthesized in muscle can be transported to the liver and converted back to
glutamate and ammonia by glutaminase. This is good because glutamate is toxic in the blood.
Thus it transfers ammonia in a non-toxic form and urea is made in the liver. Or transported to
the kidney and converted to ammonium.
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Amino Acid Metabolism_18
Alanine
Alanine can be transaminated from glutamate and pyruvate and is thus nonessential. Alanine,
like glutamine, is used as a transporter of nitrogen. This is because Glutamate (and Aspartate)
are toxic at high levels in the blood. So amino acids that are transaminated to glutamate in
peripheral tissues is combined with pyruvate to form alanine, which is nontoxic.
Alanine is then converted back to pyruvate and glutamate in the liver and glutamate yields
ammonia to make urea.
Alanine aminotransferase (ALT) is responsible for the transamination in the liver. An increase
in this enzyme level in blood (SGPT, serum glutamic pyruvic transaminase) is indicative of liver
damage.
Cysteine
Major structural amino acid that can form disulfide bonds. Key component of the tripeptide
glutathione. Nonessential since it can be formed by methionine. Free cysteine occurs as cystine
(Cys-S-S-Cys). Cystinuria (Excess cystine in the urine) due to defective kidney transporter.
Kidney stones.
Phenylalanine
Phenylketonuria (PKU) is a disease associated with the absence of phenylalanine hydroxylase,
which converts Phe to tyrosine. Autosomal recessive. Phenylalanine cannot be converted to
tyrosine so Phe builds up, which is neurotoxic. Treat by decreasing intake of Phe. Detect as
phenylpyruvate (a secondary metabolite) in urine, since Phe is readily reabsorbed. Tyrosine is
essential for these individuals.
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Amino Acid Metabolism_18
Some Important Products of Amino Acids
Cysteine is a precursor to taurine. Taurine is important for health of the retina and the heart and
is a component of bile salts.
Cysteine is also a component of glutathione, which is a general reducing agent (anti-oxidant),
especially important in the eye.
Glycine is important in creatine synthesis. Two step process in kidney and then liver. Creatine
synthesis is controlled tightly and is sensitive to ingested creatine. Creatine goes to muscle and
is used as a phosphagen. Creatine phosphate will spontaneously cyclize to form creatinine, about
1.5%/day. Creatinine is excreted in the urine.
The amount of creatinine excreted / 24 hrs is constant. In clinical chemistry, other excreted
components are ratioed against [creatinine]. Marked increase in blood [creatinine] is indicative
of kidney disorder.
Decarboxylation of histidine by histidine decarboxylase forms Histamine. A vasoactive
compound.
Glutamate is the precursor for γ-aminobutyric acid (GABA), an inhibitory neurotransmitter.
Thyroxine (thyroid hormone) is derived from iodination of tyrosine.
Dihydroxyphenylalanine (DOPA) is formed from tyrosine using tyrosinase. (Fig. 19.1)
Found in melanocytes (melanin), CNS and adrenal gland (norepinephrine and epinephrine).
Defective tyrosinase in melanocytes gives albinism. Tyrosinase positive albinism has lack of
permease for tyrosine to get into melanosomes.
Decarboxylation of DOPA produces Dopamine. A CNS neurotransmitter. Dopamine can be
converted to norepinephrine in some CNS cells. Norepinephrine is converted to epinephrine
only in the adrenal medulla.
Dopamine and norepinephrine are degraded by cytosplasmic and extracelllar Monoaminoxidase
(MAO). So physiologic action is short. Epinephrine is not affected by MAO, so its effect is
systemic.
MAO inhibitors are sometimes used to reduce blood pressure or CNS chemical imbalances. A
normal component of some foods, tyramine, (bananas, cheese) is also degraded by MAO. High
concentrations of tyramine is toxic and would accumulate in individuals taking MAO inhibitors.
So they are contraindicated.
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Amino Acid Metabolism_18
1. TYROSINASE
Parkinson’s Disease
Characterized by lack of pigment and lack of Dopamine in substantia nigra of Basal Ganglia.
Symptoms are tremor and bradykinesia (slow movements). Effects due to relative excess of
acetylcholine in basal ganglia. Treat with L-DOPA (looks like an aromatic amino acid) since
dopamine cannot cross the blood-brain barrier.
Tryptophan
Tryptophan is decarboxylated to 5-Hydroxytryptamine (serotonin), another neurotransmitter.
Which is degraded by MAO.
5-Hydroxytryptamine is a precursor for Melatonin, which is produced in the Pineal gland.
Melatonin provides seasonal and diurnal information to animals and humans.
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19. Purines, Pyrimidines and HEME (Special Products derived from Amino Acids)
Purines are made around ribose-5-Pi. If the bases were made first, they could leak out of the cell
due to high permeability.
1) ribose-5-Pi +ATP → Phosphoribosylpyrophosphate (PRPP) This is an intermediate for
purines, pyrimidines and coenzymes, e.g. NAD.
2) PRPP + 2glutamine + glycine + tetrahydrofolate + aspartate +4ATP→ IMP+ glutamate +
fumarate.
IMP (inosine monophosphate) is precursor to GMP and AMP.
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Control of Purine synthesis
1) Availability of PRPP. The enzyme making this is affected by purines and pyrimidines.
2) PRPP Transamidinase (unique to purines), inhibited 10% by AMP, 10% by GMP and 90%
by both. Thus synergistic inhibition.
3) AMP and GMP inhibit their formation from IMP.
Methotrexate and aminopterin are two drugs used in cancer treatment. They block synthesis
of Tetrahydrofolic acid from folic acid.
Purine Degradation: Continual turnover causes breakdown to uric acid. Occurs mainly in liver.
1) AMP broken to IMP and ammonia (leaves cell as glutamine). IMP is reutilized or further
degraded. Breakdown products of IMP, hypoxanthine can be salvaged back to IMP. GMP is
degraded to xanthine. Xanthine is degraded in the liver to uric acid. Xanthine and
hypoxanthine can be reused (salvaged) back to IMP.
Salvage Pathway
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GOUT is excess uric acid in blood.
Caused by eating too much DNA (meat, yeast) and/or too much reuptake of uric acid in kidney.
Buildup of uric acid in the joints due to high plasma uric acid levels. Uric acid is relatively
insoluble.
Treat with 1) Allopurinol a competitive inhibitor of xanthine oxidase, thereby slowing the
conversion of hypoxanthine and xanthine to uric acid. Xanthines are soluble, not harmful and
easily excreted. 2) Probenecid blocks uric acid reuptake in the kidney.
Defect in salvage enzyme Hypoxanthine-guanine phosphoribosyl transferase leads to
hyperuricemia, gout and mental retardation in infants. Rare genetic disorder called Lesch-
Nyhan syndrome.
Pyrimidines: Build base first, unlike Purines the intermediates are charged (so don’t leak out).
UTP and CTP Formation (Cytosol)
1) Make carbamoyl phosphate from CO2 glutamine and ATP. (see figure 20.6 in book)
2) Carbamoyl is converted to UTP (Note multiple use of amino acids)
3) UTP converted to CTP
Control
UTP feedback inhibits first step.
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Purines, Pyrimidines_19
Thymine Formation
1) CDP to dCDP
2) dCDP to dUMP
3) dUMP to dTMP Uses THFA. Methorexate is very effective (lowest concentrations needed)
in knocking out thymine synthesis.
Degradation of pyrimidines:
1) ribose broken off giving bases
2) cytosine degrades to uracil and uracil degrades to ammonia + alanine + CO2
3) Uracil can be salvaged, if not broken to ammonia and alanine
Thus pyrimidine degradation doesn’t produce any unique compounds.
HEME
Porphyrin synthesized from simple molecules, glycine and succinyl CoA. Occurs
predominantly in reticulocytes which still have organelles.
If globin is available, then Heme is continually made. If globin is not available, heme is oxidized
to hematin that inhibits first step in synthesis.
Lead inhibits Heme enzymes.
Degradation of Hemoglobin in old (120 days) RBCs takes place in the spleen. Heme separates
from globin (degraded). Heme is degraded to Biliverdin (greenish color seen in bruises). In the
spleen and liver Biliverdin is converted to Bilirubin. Bilirubin bound to albumin is transported
to the liver where it is conjugated to diglucuronate and excreted in bile as a bile pigment.
Premature infants have not developed enzymes for conjugation of bile salts, i.e., cannot form
bilirubin diglucuronide, so bile pigments accumulate and cause jaundice.
In adults jaundice (see yellow in sclera) can be caused by:
1) fragile RBCs….large amounts of bile pigments
2) hepatitis….can’t conjugate and excrete bile
3) blockage of bile duct, e.g. gallstones.
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20. Genome Organization
Genes are the functional unit of information stored in the DNA. Every cell in the organism
contains essentially the exact same genetic material, i.e. the genome.
Information flows from the genes via RNA in a process called transcription. Then from RNA
to proteins in a process called Translation.
Genetic Code
The Genetic code is a triplet of bases. Four bases in two positions would give 4x4 = 16 possible
amino acids. There are 20, so must be at least a triplet code. 4 x 4 x 4 = 64.
Using known repeating sequences of RNA bases in an in vitro protein synthesis system (ie.,
ribosomes + tRNA-a.a., etc.), it was determined that of the 64 possibilities, 61 triplets coded for
amino acids. Three were later found to be stop codons. Methionine is almost always the first
amino acid. (see Table 26.5 in book)
The code is unambiguous and degenerate, meaning that each triplet codes for only one amino
acid, but there may be more than one triplet that codes for the same amino acid. Inspection of
the code indicates that the third position is variable, but first two are not.
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Genome organization
Prokaryotes:
Phage T7 has 39,936 nucleotides, ds-linear. 45 genes organized spatio-temporally.
E. coli has 5 million base pairs in a circular chromosome of 5,000 genes about 1,000 of which
are known. Genes that code for proteins with related functions tend to be clustered together into
“operons”.
Eucaryotes:
Humans have 3.3x109 base pairs on 23 chromosomes (sperm and egg). 2x in somatic cells, i.e
46 chromosomes. 20 times the number of genes as E coli, but 1,000x more DNA! Lots of
“junk” DNA of unknown function.
Eukaryotes have mitochondrial or chloroplast DNA. In humans, m-DNA is 16,569 nucleotide
ds-circle. Each mitochondria has 5-10 copies. 13 proteins are encoded and seven specify the
subunits of NADH-Q reductase (see figure 21.3 in book)
DNA replication is made possible in chromosomes by the structure at the ends called Telomeres.
Chromosome segregation during mitosis is made possible by structures called centromeres.
Telomeres contain repeating sequences (5’TTAGGG3’). Repeat length is variable and can be
several kilobases. At the very tip, G residues are capable of forming hairpin loops through
nonstandard GG pairing. No free ends protect the chromosome from degradation by
exonucleases.
Greatest length in gametes and shorter in older individuals. Telomeres tend to get shorter with
each subsequent replication. Telomere length may be related to aging. When the telomere gets
too short, the chromosome cannot be duplicated. Maintenance of length in some cells (e.g., stem
cells) is done by an enzyme called telomerase.
Centromeres tend to be AT rich, about 130 base pairs. Forms a protein-DNA complex called
the Kinetochore that interacts with the microtubules of the mitotic spindle.
In eucaryotes, unlike prokaryotes, the genes are scattered throughout the genome with very little
apparent organization. However, there are some gene clusters analogous to bacterial operons.
For example, through gene duplication and sequence divergence the α-globin genes are located
together and the β-globin genes are located together. β-globin genes are needed sequentially
during development. A locus control region may play a role in this gene expression switching.
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Examples of Junk DNA
Repetitive DNA Sequences
Only 10% of the human Genome is required to code all the genes (100,000). What does the
remainder of the DNA do?
Dinucleotide repeats:
1) CG islands over 1kb long clustered near 5” end of gene regulatory regions.
2) AT stretches. Deletions associated with colorectal cancer.
3) CA repeats most common. Function?
Trinucleotide repeats:
CAG, 11-33 repeats normally. 40-62 found associated with Huntington’s Disease. Other
examples given in book.
SINEs (short interspersed elements). For example there are 500,000 copies of the Alu repeat
which is 300 bp long. SINEs can move around the genome. This probably leads to increased
genetic diversity, but can be problematic. For example, insertion of a SINE in a functional gene
will disrupt it. Insertional mutations by SINEs are the cause of the disease neurofibromatosis
(Elephant man syndrome)
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Genome Mapping
Traditionally this was done through linkage analysis, i.e., track down the location of an unknown
gene by associating its phenotype with a known gene. Modern molecular biology can determine
the sequence of the linked area of chromosome. Then the coding and regulatory regions can be
determined. Example, cystic fibrosis gene found this way. Not just one mutation, but several
with varying effects on the phenotype.
Restriction Fragment-Length Polymorphisms (RFLPs). Everyone has DNA polymorphisms,
which are slight differences in the DNA sequences among us. Most occur in non-coding regions
since most of the DNA is non-coding. These polymorphisms can be used as markers, especially
when they are linked to defective genes. Some polymorphisms are within the gene, i.e. a
mutation. This can be used for prenatal analysis of known genetic diseases using Southern
blotting.
An example of restriction enzyme digestion of normal and sickle cell β-globin gene is shown
below.
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21. DNA Replication
Function: replicate the genome within a short time to prepare for cell division.
Requirements: speed and error detection with repair.
In humans, S phase to replicate 3x109 bp is 8 hours, while bacteria take 30 min. to replicate
5x106. So bacteria are slower/bp.
Conservative or semi-conservative?
Conservative replication means that two new daughter complementary strands are made and the
parent double helix is conserved. Semi-conservative means that one new daughter strand is
synthesized complementary to one parent strand. Meselson and Stahl used heavy and light
nitrogen labeling to decide between the two.
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Enzymology of DNA Replication
Double strand with single strand RNA primer is needed. DNA polymerase adds dNTP to 3’ –
OH group. PPi is split off. Thus energy comes from the high energy triphosphate of the dNTP.
Only 5’ to 3’ DNA Polymerases.
Where does replication begin and does it go in one direction or two?
In E. coli there is one unique origin that is seen by pulse labeling with radioactive thymidine.
In eukaroyotes there are many origins of replication seen on each chromosome.
Only 5’ to 3’ DNA polymerases have been found. How can synthesis go in both directions, if
DNA polymerase can go only one way?
Short pulse labeling revealed long fragments and many short fragments (Okazaki fragments).
Thus one strand proceeds 5’ to 3’ continuously (called the leading strand) and the other strand
proceeds 5’ to 3’ discontinuously (called the lagging strand). This combination gives net
movement of a “replication fork” in one direction.
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DNA Pol III is responsible for DNA replication at the replication fork. Pol III has the highest
rate of elongation and best processivity (ability to elongate without falling off). β-subunit of Pol
III acts as a clamp around the template strand.
If a base mismatch is incorporated, the DNA Pol will stop, back up and excise the nucleotide.
This requires exonuclease activity. If a mismatch is missed it must be recognized by other DNA
repair mechanisms. Together, less than 1x10-9 errors /bp. 3x109 bp so 1-3 errors could occur!
Replication fork is maintained by a Replisome Protein Complex.
Function:
1) Unwind/denature DNA (helicase)
2) Prevent renaturation (single strand binding protein or SSB)
3) Prevent supercoiling (Topoisomerase)
4) Provide RNA primer (DNA primase)
5) Remove RNA primer and fill (DNA Pol II)
6) Tie together DNA fragments (DNA Ligase)
Origins of Replication
In eukaryotes there are origins every 105 bp, minimum number of origins needed to complete
synthesis of entire genome in 8 hours is an origin every 2 x106 bp. Some origins are started
earlier than others during S phase. Unlike prokaryotes, sequences are not specific, so Origins
may be controlled by binding proteins.
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22. DNA Repair and Recombination
Because of the immensity of the genome, mistakes are inevitable. A repair system is needed to
remove mistakes. The repair system is very efficient however, ~1 base change /109 bp is passed
on.
Mutations are caused by DNA damage.
Mechanisms:
1) Spontaneous Depurination of G or A to deoxyribose through hydrolysis of N-glycosyl
linkage. 1000/109 bp per day!
2) Deamination of cytosine to uracil that could become an A-T bp if not corrected before DNA
synthesis.
3) UV light and ionizing radiation can form thymine dimmers. These prevent protein binding
and inhibit transcription.
4) Mutagens. Certain chemical that react with bases and alter them. For example, nitrites are
deaminating and dimethylsulfate adds methyl groups. (screen with the Ames test, measures
the mutation frequency of a test gene in the presence of test chemical).
Mutations
1) Point Mutations (3 possible results)
a) no effect due to degeneracy of the code
b) missense a.a. substitution: no effect, acceptable or unacceptable
c) non-sense codon, i.e. stop
2) Frame shift mutation addition (e.g., intercalating agents) or deletion of a base leads to a
garbled message
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The acceptable mutations could be better, in which case they may be selected for. This is the
process of Natural Selection.
Repair Mechanisms
Damage is usually limited to one strand. Four basic steps:
Recognize, remove, resynthsize and ligate.
1) Excision repair (bulky lesion repair): Recognized as a structural abnormality in the DNA
backbone. Example removing thymine dimers. Xeroderma pigmentosa is a disease with
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multiple defects in repair enzymes for thymine dimers. These people are predisposed to skin
cancer.
2) Mismatch Repair. In prokarotes, recognition of wrong base on new strand depends on the
fact that new bases are not methylated. Mismatch is recognized and unmethylated base is
removed. Human analog to E. coli repair genes have been found and defective ones are
associated with colon cancer.
3) Base-Excision repair (Strange base or missing base). For example, repair depurination events
(no base) and repair deamination events that have lost the base following removal by DNA
glycosylases. DNA glycosylases are enzymes that recognize inappropriate bases such as
uracil, hypoxanthine, xanthine caused by deamination of cytosine, adenine and guanine,
respectively and remove the base.
4) Direct repair. methylguanine made by alkylating agent can pair with thymine.
Methylguanine DNA methyltransferase removes methyl group and thus “directly repairs”
mutation.
RECOMBINATION
Homologous (general) and Site-specific.
In eukaryotes Homologous recombination occurs during meiosis just after Chromosome
duplication (4N). Cross-over frequencies help in gene mapping through linkage analysis.
Site Specific Recombination occurs at specific sequences. This is illustrated by
immunoglobulin genes.
In order to be able to respond to the immense number of possible antigens, each developing
lymphocyte makes a unique antibody. Heavy and light chains are made up of variable (V) and
constant (D and J) polypeptide regions. Imprecise rearrangements among 1000 V regions, 12 D
and 4 J regions during lymphocyte development give immense antibody diversity.
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Gene Knock-outs
One way to fully understand the function of a gene and all the ramifications of its expression is
to eliminate (knock-out) the gene from the animal. This can be done in the mouse by targeting a
gene to insert into the gene of interest to make it non-functional.
1) Construct a vector that has sequences flanking the target gene that are the same as normal,
but in which an antibiotic resistance gene has been inserted into the target gene and a herpes
simplex virus thymidine knase (tk) gene has been added. The antibiotic resistance gene
(Neo) will allow cells to survive in the presence of the antibiotic genticin, however if the tk
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gene is expressed, thymidine kinase will phosphorylate thymidine in a way that makes the
cells sensitive to drugs (anti-Herpes drugs) that inhibit DNA synthesis, (e.g., gangciclovir).
2) Insert the vector into mouse embryonic stem cells in culture.
3) Select cells that can survive in the antibiotic (antibiotic resistance gene inserted) +
gangciclovir (tk gene is not expressed).
Only those cells with double homologous recombination between Neo gene and HSV-tk are
resistant to both genticin and gangciclovir.
4) Grow up stem cells and implant in pseudopregnant mouse. Some stem cells of chimeric
mouse will have knockout. Inbreeding of these mice can yield homozygous knock out
mouse.
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RNA Synthesis_23 V515
23. RNA Synthesis
Transcription: Process of reading DNA code into a complementary RNA molecule.
Differences between RNA and DNA synthesis:
1) ribonucleotides rather than deoxyribonucleotides
2) uracil replaces thymine
3) does not require a pre-existing primer
4) RNA synthesis is very selective
Four Major Classes of RNA
1) Messenger 5% very complex
2) ribosomal 80% four types, so sequence complexity is low
3) transfer RNA, 50 types
4) small nuclear RNA, 10 types
A gene is a segment of DNA that specifies and regulates a transcription unit. Transcription is
initiated at the 5’ end of the gene in the region called a promoter. Regulatory sequences are
found upstream from the promoter. (see Figure 24.1)
Transcription is produced by RNA polymerase holoenzyme, A Multisubunit enzyme.
In bacteria there are about 6 subunits. One subunit’s job is to recognize the promoter. Once the
holoenzyme binds and elongation of RNA begins, this promoter subunit dissociates.
There is no exonuclease proofreading activity in RNA polymerase, so error rates are higher. No
consequence to genetic material.
Eukaryotes have 3 RNA polymerases: RNA Pol I for rRNA, Pol II for mRNA, and Pol III for
tRNA and snRNA. A separate nuclear encoded mtRNA Pol is also made.
RNA synthesis: Initiation, elongation and termination.
Template strand is read and nontemplate strand or “coding” strand has the same sequence as the
nascent RNA.
Initiation (see diagram next page)
In bacteria there are two highly conserved DNA sequences within the promoter: One is centered
35 bases upstream of the initiation site and the other is 10 bases upstream. RNA Pol slides along
DNA to the 35 site and binds, called the closed complex. It then slides to the –10 site where it
unwinds the DNA, called the open complex. Promoter sequences are often common, e.g.
TATAA and CAAT. Whether the promoter sequence is available for binding is dependent on
upstream regulatory sequences and their associated binding proteins.
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Elongation
5’ to 3’ direction, energy derived from high energy phosphate NTPs. Multiple RNA Pols latch on
simultaneously so many copies are being made. Speed is 30 bases per sec, so 2kb in about a
minute. Base pairing of RNA to DNA occurs only in the Elongation Bubble (15 bases).
Topoisomerases relieve torsional stress.
Termination
Two methods: protein dependent and independent. Both methods utilize a hairpin loop coded at
the 3’ end. After making the loop, RNA Pol pauses. A ρ protein comes in and causes
DNA/RNA disassociation. Protein independent uses a further string of A-U base pairs that
destabilize when RNA Pol pauses.
Some antibiotics work at various points in transcription or translation. Actinomycin D blocks
elongation at low concentration in both pro- and eukaryotes. Nice tool, but not used clinically
because it would be toxic to the host. Rifamycin from Streptomyces (rifampicin used clinically
is analog) blocks initiation. This is clinically useful because it only works in prokaryotes.
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24. RNA Processing
Bacterial genes are transcribed continuously, sometimes multiple genes together, with no “junk”
in between.
In contrast, eukaryotic hnRNA needs to be highly processed to remove introns, splice exons and
add other bases.
Overview of RNA processing.
First Step in Processing adds 5’ methyl Guanosine cap and a 3’ polyadenylated tail.
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The cap is added shortly after RNA synthesis by enzymes associated with the RNA Pol II
holenzyme. Needed for 1) initiation of protein synthesis and 2) protects 5’ end from
exonucleases.
Second, the sequence AAUAA is found at the end of most mRNA. This serves as a binding site
for two proteins that first cleave the mRNA 20 bases downstream and then add up to 200 A
residues.
Third, Long Poly A tails give the mRNA stability and enhance translational efficiency.
Splicing of exons
In fungi and yeast introns are self-splicing. The RNA structure in self-splicing mechanisms is
what gives the RNA intron “enzymatic” activity, i.e. denaturation inhibits splicing activity. This
activity has been termed Ribozyme.
Splicesome (Protein) Mediated: Predominant form in higher eukaryotes. Small nuclear RNA
(snRNA) molecules and protein cofactors that form small nucleoproteins (snRNPs), called
snurps, are needed.
GU-AG residues are conserved at the intron boundaries, however variations in remaining intron
sequences are possible. These variations could give rise to variable protein binding. Gives
flexibility and a point of possible regulation for alternative splicing. Imagine that a gene has 6
exons, but only 5 are used to make the functional protein. The sixth is skipped. Thus there are 6
different “splice variants” that are possible.
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rRNA is transcribed by RNA Pol I in a long string with introns. The rRNA sequences are
methylated and the introns are degraded by ribonucleases.
tRNA processing requires several steps:
1) Cleave tRNA out from large precursor and remove introns if present.
2) Add CCA charging sequence
3) Modify several bases by reduction, methylation and transamination. Function is to modify
strength of codon recognition and along with anti-codon help specify amino acid that’s
charged.
RNA Degradation
Steady-State life of mRNA is determined by the rate of transcription and the rate of decay. RNA
decay involves eating away the poly A tail.
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Example of regulation of poly A tail life: Iron responsive element binding protein (IRE-BP)
binds iron if available. If iron is not available, then IRE-BP binds stem-loop structure of
transferrin receptor mRNA and stabilizes it. If you have more transferrin receptor, then the cell
can pull in more transferrin, which has bound iron.
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25. Protein Synthesis
Processed mRNA moves to the ribosomes to be translated into a polypeptide strand. The a.a.
sequence is determined by a triplet code of bases. The polarity (i.e., amino vs carboxylic acid
end) is determined by the 5’ to 3’ sequence of bases. tRNA acts as the adapter molecule between
mRNA and amino acids. The genetic code determines which tRNA (i.e. which a.a.) is inserted
into the ribosome. The third class of RNA, rRNA, takes an active role in protein synthesis.
Predicting open reading frame
So where is the polypeptide in a sea of bases? Where does it start and where does it finish?
1) There are three possible reading frames. Need an AUG start and one of the 3 stop codons. In
between should be something of reasonable length. See figure 26.4 in book
2) Use cDNA sequence. Computer algorithms can find open reading frame of 100 codons or
more that would approximate a reasonable size protein.
Exceptions to the Central Dogma (DNA to mRNA to proteins through strict base pairing that is
conserved)
1) Translational frameshifting: Example in virus where gag protein (needed for viral
packaging) terminates at a UAG. However if ribosome “stutters” or skips over one base at
stop codon, a longer mRNA is made encoding both gag and pol (reverse transcriptase). The
fusion protein is cleaved by an endoprotease. This happens 5% of the time, which reflects
the relative need.
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2) RNA editing. Postranscriptional change of an RNA base. Example: Apo B, lipid binding
protein. In intestinal cells, a cytidine deaminase converts CAA (glu) to UAA (stop) making a
shorter message than found in the liver. Both however are functional.
3) Protein splicing. Example in yeast. Predict ~119 kD for an ATPase from the open reading
frame. Find it is only 69 kD. 119kD was synthesized and then paired down. Insulin is also
synthesized as a long polypeptide that is cleaved and spliced together.
Properties of tRNA, the Molecular Adaptor Molecule
1) the anticodon and the amino acid are physically separated.
2) Many of the bases are post-transcriptionally modified, some are uniform across tRNAs others
are unique.
3) There are four intrastrand RNA-RNA helices formed by standard and nonstandard base
pairing.
4) The 3’ end contains CCA, which is linked to the amino acid.
The anticodon sequence and the variation in modified bases in the backbone are sufficiently
different to distinguish the tRNAs from each other.
Amino acids are charged onto tRNA by 20 different aminoacyl-tRNA synthtases. Each
synthetase is bifunctional in that it can distinguish each amino acid and the appropriate tRNA(s),
i.e. more than one tRNA can be used for some amino acids.
Inspection of the genetic code indicates that the third position is variable, but first two are not.
Francis Crick came up with the Wobble hypothesis. It says that the first two bases of the anti-
codon follow strick Watson-Crick base pairing, but the third postion can have nonconvetional
pairing. In fact, the third position on tRNA often is inosine. Thus a single tRNA could
recognize more than one codon. Ribosome binding assays confirmed the hypothesis.
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Initiation (see fig. 26.10 in book)
1) assembly of the initiation complex: various protein factors and 30S ribosomal subunit come
together at a “translational start site”, (Shine-Dalgarno sequence) just upstream from AUG
start codon, sometimes called the 5’-UTR (untranslated region).
2) fMet-tRNA + another initiation factor + GTP binds to AUG.
3) GTP hydrolyzes, initiation factors dissociate and 50S subunit binds.
Initiation is similar in eukayotes.
fmet-tRNA is only used for initiation. Another met-tRNA exists. fMet or just the formyl group
are later removed. In eukaryotes fmet isn’t used , but there are two met-tRNAs.
Elongation (figure 26.11-26.13 in book)
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1) 50S ribosome has two pockets or sites. ‘P” site for peptidyl and “A” site for aminoacyl or
acceptor. The “A” site is then filled by the appropriate charged tRNA.
2) α-amino group of the amino acid in the “A” site attacks the carbonyl group of the amino acid
in the “P” site, giving a dipeptidyl-tRNA in the “A” site and an empty tRNA in the “P” site.
Peptidyl transferase is catalyzed by ribozyme activity!
3) Ribosome translocates one codon in the 3’ direction. The dipeptidyl-tRNA is now in the “P”
site.
Termination (figure 26.14 in book)
When ribosome reaches a stop codon, no tRNA can enter, however termination factors can bind
and catalyze release of polypeptide, tRNA and ribosome subunits.
Read-through of stop codon can occur in translational frameshifting or if there were a mutant
tRNA that recognizes a stop codon. Actually found in bacteria. Function?
Inhibitors of Protein Synthesis
See antibiotics in table 26.1
Many antibiotics work by interfering with protein synthesis at the ribosome. In order for them to
have therapeutic value, they must be more potent against bacterial ribosomes than against
eukaryotic ribosomes. For example, tetraycline is good against prokaryotes, but does not affect
eukaryotes. On the other hand cycloheximide can interfere in both pro- and eukaryotes.
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26. Protein Targeting and Turnover
How do proteins know where to go? They contain a signal peptide (continuous) or signal patch
sequence (discontinuous) that directs them to their destination. (see Figure 27.1 in book)
Signal peptide sequences are often found at the amino terminal end. While signal patches are
discontinuous in the linear peptide, folding brings them together.
Nuclear Localization
Cells that are actively transcribing or dividing have more nuclear pores. For example, in cell
synthesizing DNA, 106 histone molecules must be transported from cytosol to nucleus every 3
minutes.
Nuclear proteins extracted from cells and then microinjected back into the cytosol will move
back to the nucleus. This is due to nuclear localization sequences.
1) Located anywhere on protein
2) 4 to 8 amino acids
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3) rich in lys, arg and usually contain proline
Mitochondrial Localization
Mitochondrial proteins are taken up from the cytosol in a few minutes. They have an amino
terminal SP of 20-80 amino acids that is rapidly removed after import by a signal peptidase in
the mitochondrial matrix.
These SPs form an amphipathic α-helical structure with positively charged amino acids on one
side of the helix and hydrophobic residues clustered on the opposite side.
1) Need SP
2) Need hsp70 chaperone protein (keeps protein unwound)
3) need ATP
4) need electrochemical gradient
Peroxisomes: Oxidative reactions used to detoxify alcohols, phenols, formaldehyde, breakdown
fatty acids (β-oxidation). Prominent in liver and kidney.
Short signal sequence. Defect leads to empty peroxisomes, called Zellweger syndrome. Die
shortly after birth.
ER Targeting
Best characterized SP is for the ER. Approx. 10 hydrophobic a.a at amino terminal end. If
present, polypeptide is secreted into ER. If ER SP is not present, translation continues on
cytosolic ribosomes.
These proteins will either remain in ER, move to Golgi and/or be secreted to membranes of
lysosomes. (see figure 27.3 in book)
1) SP sequence, very hydrophobic, is started on nascent polypeptide
2) This is recognized by an ER signal recognition particle (SRP). SRP is a riboprotein complex
that binds to both SP and to ribosome. Protein synthesis pauses.
3) Protein synthesis resumes with polypeptide feeding through a channel into the lumen of the
ER.
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4) SP is either cleaved, remains for further direction or anchors protein in ER membrane.
In ER many proteins are modified by glycosylation adding 14-residue oligosaccharides. Proteins
either remain in ERor move to the Golgi
Golgi
1) Formation of proteoglycans (long polysaccharides with short polypeptide)
2) Processing of polyproteins by proteolysis to form more than one mature protein, e.g. insulin.
3) Distribute to: plasma membrane, lysosomes or secretory vesicles.
Protein Turnover
Two mechanisms: Proteasome directed and lysosome directed.
Need to remove:
1) unassembled proteins
2) misfolded, denatured, abnormal amino acid containing proteins
3) regulatory proteins
1. Proteins in the cytosol are degraded by Proteasomes. Central cylinder formed from multiple
distinct proteases, whose active sites are thought to face the inner chamber. Proteasomes
recognize proteins for degradation that are coated with a protein called ubiquitin.
Each protein has a ubiquitin directed degradation signal. There are many ubiquitin associating
enzymes specific for each sequence. Sequences could be buried and when exposed following
damage, the protein is ubiquitinated. (see figure 27.6)
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Example: Cyclin turnover, proteins needed for cell cycling are degraded by protein degradation.
Possibly they are temporarily protected by other transient cofactors.
Stability of a protein could be influenced by its N-terminal amino acid, called the N-end rule.
The destabilizing amino acids (there are 8) are rare in cytoplasmic proteins, but are found in
those that are compartmentalized. Thus it may be that wayward compartment proteins are
rapidly degraded.
2. Degradation of Endocytosed proteins and organelles
As mentioned previously, Lysosomes act as protein degradation sites for endocytosed material
and for autophagy of old and damaged organelles, like mitochondria.
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27. Biotechnology
Cloning: Genomic DNA and cDNA
1) Create cDNA library: Make cDNA by reverse transcription of mRNA. Ligate to plasmid and
insert into bacteria. Each colony will grow up that makes many copies of unique cDNA.
2) Screen library: need an oligonucleotide probe. Probe sequence is based on:
a) amino acid sequence or
b) homology with other similar types of proteins
3) Isolate colony, retrieve cDNA from plasmid, sequence and determine amino acid sequence.
To get the whole gene (coding sequence plus all the upstream regulatory sequences and all the
introns) create a Genomic library.
1) chop up DNA from cells and insert into plasmids
2) screen bacterial colonies for expression of DNA of interest by probing with your cDNA
Special Uses of DNA Polymerases
1) Reverse transcriptase discovered in retrovirues. Makes cDNA from RNA template. Used in
molecular biology to make cDNA from mRNA.
2) Taq Polymerase is a high temperature resistant polymerase that is used in PCR.
PCR (Polymerase Chain reaction) (See figure 22.12 in book)
Process: Amplify DNA or RNA samples over a million fold.
Uses: 1) detect latent viruses
2) prenatal screening from chorionic villus or amniotic cells
3) Forensic identification
4) Make cDNA probes
Start with cDNA from cells of interest. Design upstream and downstream primers (~20
nucleotides) that are complementary to + strand and – strand, respectively. The distance between
the primers should be 300 bp or greater.
Denature DNA at 94°C, 30sec; add primers bind 65°C, DNA synthesis 72°C (optimum for Taq).
What you get primarily is DNA synthesized between the primers. Do this for 25-40 cycles to
amplify this DNA.
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Site-Directed Mutagenesis
Delete or insert bases in sequences and see how the protein functions.
Screen colonies with labeled oligonucleotide probe for mutant sequence.
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28. Gene regulation in Prokaryotes
Regulation occurs at the level of transcription. This is the most energy efficient way.
Typically there exist upstream regulatory regions that are not transcribed. The regulatory region
controls the rates of transcriptional initiation at the promoter. How this happens is not entirely
clear.
Cytoplasmic regulatory factors (effector molecules) need a mechanism for interacting with the
genome. Transcription factors are proteins that bind to the specific regulatory regions.
Transcription factors interact directly or indirectly with the transcriptional initiation complex.
Transcriptional factors can be activators or repressors. The specific sequence within the
regulatory region that binds the transcription factor is called the response element.
Genes with low basal levels of transcription are often responsive to activators, whereas genes
with high basal rates of transcription are responsive to repressors. Transcription goes up and
down, i.e. respond to activators or repressors, depending on the cellular environment.
Housekeeping genes are often constituitively expressed and do not respond to activators or
repressors.
Changes in the cellular environment are often transduced through the action of effector
molecules. Effector molecules can bind to transcription factors and alter binding. Examples,
cAMP, metabolic products, steroids, phosphorylated intermediates.
General Mechanisms of regulation
1) direct induction
2) deinduction
3) direct repression
4) derepression
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The lac Operon (example of Derepression)
Function: Produce enzymes needed for metabolism of lactose. Lactose is a disaccharide, which
when cleaved by β-galactosidase produces glucose and galactose. Bacteria prefer glucose, so
when glucose is available, a mechanism for turning off the lac operon would be most efficient.
Three genes: lacZ (β-galactosidase), lacY (permease), lacA(acetylase).
These genes are transcribed in one shot and translated as independent polypeptides. This is
called polycistronic mRNA.
Just upstream from these genes are the operator, promoter and lac represor gene (lacI).
Lactose induces lac Operon transcription. A synthetic non-hydrolyzable analog (IPTG) does
the same.
Genetic mutations were used initially to understand mechanisms of regulation of lac Operon.
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First mutation caused the gene to be always on. This mutation mapped to a region of the lac
promoter called the lac operator. Called constituitive mutant.
Second mutant was also constituitive. This mapped to the lacI gene. The protein product was
found to be a repressor and the mutation decreased its DNA binding activity.
Mechanisms of Regulation of lac operon
1) In the absence of inducer (i.e. lactose) the repressor binds very tightly to the lac operator and
prevents theRNA polymerase initiation complex from moving.
2) Inducer (lactose or IPTG) interacts directly with the repressor and reduces its binding activity
for the lac operator. So the inducer works by transcriptional de-repression. DNA
footprinting (see book for explanation of footprinting) indicate that RNA polymerase and
LacI repressor binding is mutually exclusive, i.e. the sequences they bind overlap.
3) In the presence of lactose and glucose, lac transcription is low even though repressor is not
bound.
4) However, when glucose is low and lactose is available, transcription increases. This is due to
another effector molecule called CAP-cAMP (catabolic gene-activator protein cyclic
AMP). This binds just upstream from promoter and binds directly to E. coli RNA
polymerase.
5) Glucose transport leads to deactivation of adenyl cyclase and thus lowers cAMP.
Conversely, low glucose keeps cAMP high, which activates CAP, allowing it to bind.
6) When both glucose and lactose are low, the operon is turned off.
Gene regulation in Eukaryotes Chapter 29
Nucleoprotein complexes
Chromosome Functions
1) Storage and transmission of genetic information.
2) Expression of genetic information
3) Maintenance of genetic information, i.e. DNA repair.
4) Recombination of genetic information.
Major Concept: All these functions are controlled by protein interactions.
Most common chromatin protein are the histones. (Chromatin = DNA + proteins, predominatly
histones). Histones are very basic due to high content of basic amino acids lysine and arginine.
They have a central structured domain and random coil ends for lying in major and minor
grooves of double-stranded DNA. Post-translational modification of histones may affect gene
expression.
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Phophorylation of H1 is associated with condensation of chromatin, i.e. inactive.
Acetylation of H3 and H4 is associated with enhanced transcription, etc. see book
Nucleosomes are structures that have DNA wrapped around histones. Phosphate groups of DNA
are neutralized by basic amino acids of histones. Most cellular DNA is in a nucleosome
structure.
Inactive regions are coiled nucleosomes called solenoids. Major Concept is that tightly wound
DNA is not transcribed.
Transcriptional units are often arranged in physical domains. Each domain of DNA is 50-100 kb
with the ends anchored to a large stable protein structure called the nuclear matrix or scaffold.
Accessibility to DNAses is a measure of exposure and thus activity.
Euchromatin or active chromatin is chopped up by DNAses, while heterochromation is not.
Three regions: 1. resistant, 2. Sensitive (10x more than 1) and 3. Hypersensitive, like free DNA.
Hypersensitive regions are small and scattered and tend to flank sensitive regions, may be sites
for control of transcription. These sequences will bind to transcription control proteins.
Sequence-specific DNA-Binding proteins have specialized functions like maintaining telomeres
or regulating transcription. Three major types identified. Helix-turn-helix motif, zinc-finger
motif and leucine –zipper motif. Proteins can be isolated by gel-mobility shift assay (see book).
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Differences from prokaryotes:
1) All genes are not used in a eukaryotic cell
2) DNA condensation is highly variable
3) Post-transcriptional steps are subject to regulation
4) Large numbers of proteins used to regulate genes.
5) Response elements affecting transcription can be close or far away.
STEROID/NUCLEAR RECEPTORS
The glucocorticoid receptor is present in the cytoplasm. It binds cortisol and goes to the
nucleus. Dimers of the receptor bind to transcription elements and genes are transcribed.
Receptor is recycled and refolded by a chaperone protein hsp90 (heat shock protein 90).
The glucocorticoid receptor is in a superfamily of steroid/nuclear receptors with some homology
in their DNA binding domains
Nuclear receptors for retinoic acid, RAR (cell development, proliferation, increased glycoprotein
synthesis); thyroid,TR; and vitamin D, VDR (promotes calcium uptake in gut) all have the same
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binding sequence, which is also the same as that for estrogen. However, the estrogen receptor
forms homodimers.
From left to right shown below are cortisol, estradiol, testosterone, Vitamin D (top)
thryroxine and retinoic acid (bottom).
estradiol testosterone
Vitamin D
cortisol
thyroxine
Retinoic acid
D
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EXTRACELLULAR SIGNALING
Phosphorylation cascades induced at the membrane are cascaded onto cytoplasmic kinases that
can phosphorylate nuclear transcription factors.
CREB, cAMP-response-element binding protein. Protein kinase A is activated by cAMP.
Dissociation of the regulatory subunit allows the catalytic subunit to enter the nucleus where it
phosphorylates CREB and CREB binding protein.
Growth factor (EGF, insulin like growth factor, γ-interferon, etc.) signaling transmitted by
tyrosine kinase activation. Jak and STAT family of transcription factors are phosphorylated by
tyrosine kinases.
γ-interferon activates Jak1 and Jak2 kinases, Jak1 and Jak2 then phosphorylate STAT1-α and
STAT1-β. These go the the nucleus and bind to γ-interferon response element.
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29. Animal Viruses
Viruses are infectious particles that rely on cellular machinery for replication. The fate of the
cell is: 1) death or lysis, 2) latent infection, 3) induced proliferation.
Parts (Given all the parts, viruses can self-assemble in the cell cytoplasm)
Nucleic acid (RNA or DNA)
Protein coat called the capsid
Enveloped viruses have lipid membrane envelope.
Glycoprotein spikes (vaccines are generally developed against spike proteins).
A) Adsorption, then Penetration:
Endocytosis at specific receptor is the most common way for viruses to get into cells. Some
viruses use less specific means.
B) Fusion with endosome
C) Escape from fusion with lysosome
E.g. in semilike forest virus the low pH of endosomes causes glycoproteins of viral
envelope to fuse with endosome membrane, resulting in release of nucleocapsid.
E) Make Virions
Capsid disassembles releasing naked nucleic acid. This is eventually translated by cell to make
viral proteins.
Capsid is made on free ribosomes and associates with nucleic acid in cytoplasm.
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Envelope proteins are made via ER and Golgi, which fuse with plasma membrane.
Coat or capsid proteins used to make vaccines. Vaccines become ineffective due to mutations
in capsid and coat proteins. Example, new influenza vaccines made each year.
Classification:
Viral nucleic acid- first replication product
1) RNA-RNA (+) or (-) strand
2) DNA-DNA single or double stranded
3) DNA-RNA
4) RNA-DNA
RNA-RNA
Polio (Rhinovirus, rubella) is a (+) strand RNA virus. (+) strand is mRNA. Made into a single
polypeptide that is cut up into capsid and processing proteins. Processing proteins include a
replicase to make (-) RNA template and then make many new copies of (+) strand for
packaging. Polio is effective at stopping host translation. It produces a protease that cleaves
host initiation factor.
Influenza (rabies) is a (-) strand virus. RNA transcriptase contained in virion makes (+) strand.
Hepatitis A causes infectious hepatitis.
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DNA-DNA
Large variation in genome size. Small DNA viruses require cells that are replicating since they
do not encode a DNA polymerase.
SV40 best studied. Circular double stranded DNA. SV40 can transform cells, (i.e., cause them
to proliferate).
Early promoter makes T antigen (transforming protein). T antigen is multifunctional. It is
required for SV40 replication and late transcription.
Transformation properties are due to T antigen binding to Rb and p53 proteins of host.
Normally p53 and Rb are required to prevent cell replication. Binding inactivates them, leading
to uncontrolled proliferation.
Herpes is a large DNA-DNA virus. Cause cold sores and genital blisters. Antiviral drugs are
nucleotide analogs (gancyclovir, acyclovir, iododeoxyuridine). Herpes thymidine kinase will
preferentially phosphorylate analogs, incorporate them into its DNA and cause DNA replication
to stop.
Herpes can integrate into the host genome. Causes no problem, but virus is latent and can be
reactivated. Example: Chicken pox (Herpes Zoster)
Adenovirus (colds respiratory infections). Produces E1A protein, which is an effective RNA
pol II initiation complex cofactor. Thus it can stimulate both host and viral transcription.
DNA-RNA Viruses
Hepatitis B causes serum or chronic hepatitis
1) DNA converted to RNA by cellular polymerase.
2) RNA is used as a template by HBV encoded reverse transcriptase to synthesize new viral
DNA.
3) HBV X protein modulates host gene expression that can stimulate phosphorylation cascades.
4) HBV genome can integrate into host which could inactivate genes by disruption or activate
by way of its transcriptional regulatory sequences.
#3 and #4 thought to be involved in producing liver cancer.
RNA-DNA or Retroviruses
1) two copies of (+) RNA and two molecules of reverse transcriptase
2) RT makes RNA-DNA hybrid which is then converted to DNA duplex
3) DNA duplex integrates into host. Called provirus.
4) Viral RNA is transcribed.
HIV has a predilection for CD4 T cells (helper cells).
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Treat with nucleotide analogs that block reverse transcriptase, e.g. AZT. Lack of proofreading
on reverse transcriptase leads to high mutation rate, which limits use of these drugs.
Protease inhibitors: Viral proteins need to be processed by proteases to be functional. These
new inhibitors are designed to inactivate them.
Gene Therapy: Virus vectors can be used to insert good genes into defective cells.
Adeno virus used in respiratory gene replacement, e.e. CFTR.
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Induction of Latent Virus
Lambda phage:
1) Phage incorporates into bacterial chromosome
2) Remains dormant due to “r” protein that represses transcription of cro and other gene
products.
3) Lytic response starts by sensing cell damaging substances, e.g. UV light →ss DNA
4) ss-DNA activates a bacterial protease (recA) that degrades r protein, so cro is transcribed.
5) Cro turns off transcription from repressor promoter, so no more repressor protein.
6) Other lytic proteins produced. Irreversible.
Herpes is a latent virus. Resides in trigeminal ganglion.
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30. Oncogenes and Human Cancer
Cancer due to perturbations in regulatory mechanisms.
Could be loss of function (DNA repair enzymes, cell cycle control) or gain of function
(mutations in growth-stimulatory proteins).
Oncogenes are cancer causing genes that have mutations leading to loss or gain of function. The
normal unmutated genes are called proto-oncogenes.
Cancer is highly correlated to DNA mutations
1) Incidence increases exponentially with age, suggesting accumulation of unrepaired somatic
mutations.
2) Strong correlation between potency of DNA-damaging agents causing mutations in bacteria
(mutagens) and their ability to cause cancer in animals. (Ames test)
3) Karyotyping of tumor cells shows gross chromosome rearrangements, e.g. rearrangement
between 9 and 22 give Philadelphia chromosome in myelogenous luekemia.
Natural History
Most cells divide during development, reach a predetermined fate and then terminally
differentiate (stop dividing). Enter a G0 phase in the cell cycle.
By altering cell cycle control, cells can dedifferentiate and divide to form benign growths.
Example: papilloma wart from virus infection.
UV light induced mutations can lead to benign skin growths in basal cells of dermis or in
melanocytes. Given the increased cell divisions, there is a greater chance for further mutations
and some cells may acquire mutations that give them invasive properties (secretion of proteases):
attract vascular endothelium and breakdown basement membrane and connective tissue =
Malingnancy. Example, basal cell carcinoma (only locally invasive), melanoma (metatasizes).
Some cancers can be promoted by normal hormones, e.g. estrogen (breast cancer) testosterone
(prostate cancer). The combined effect of an initial DNA mutation + altered gene expression
due to promoter could accelerate development of malignant growth.
Assay for cancer cells
Immortalized cells = cells that grow, but retain contact inhibition and do not form tumors.
Transformed cells = cells that do not stop growing when confluent, form tumors in animals,
grow in soft agar, grow to high density, and have fewer growth factor requirements.
Oncogenic Retroviruses in animals gave first clue about oncogenes.
Atypical viral sequences were found to resemble cellular protein kinases. DNA transfection of
these sequences alone imparted transformation. The retroviral oncogene was either truncated or
contained small deletions or point mutations, most likely in regulatory regions. Thus these were
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‘turned on” or dominant oncogenes since they caused transformation even in the presence of the
normal proto-oncogene.
Retroviruses may have “captured” these oncogenes through host mRNA that is reverse
transcribed by viral RT to cDNA that is incorporated into the viral genome.
First human oncogene found was ras.
Human tumor DNA was randomly sheared and used to transform mouse 3T3 fibroblasts.
Human DNA was retrieved from the transformed mouse cells. DNA was sequenced and had
homology to retroviral oncogenes. ras codes for a GTP-binding protein in a signal transduction
pathway.
Ras is stimulated by growth factors. This leads to displacement of GDP by GTP, the “activated”
Ras. Ras has GTPase activity to return it to inactive state.
Mutation in GTPase then will leave ras permanently “on”. Active ras phosphorylates Raf which
phosphorylates MAP kinase kinase that eventually lead to phosphorylation of transcription
factors Fos and Jun causing uncontrolled growth. Thus mutations in Fos or Jun could do the
same, so they are oncogenes as well.
Tumor-Suppressor genes
Loss of activity causes cell division when it is not supposed to.
Retinoblastoma is a childhood ocular tumor of undifferentiated retinal neurons. In the
hereditary form, multiple tumors formed in both eyes. In the acquired form, usually one tumor
in one eye is found.
Hereditary needs only one-hit to be homozygous. Spontaneous form needs two-hits to be
malignant.
Rb gene codes a cell-cycle control protein that prevents cells from replicating. When
phosphorylated it binds to DNA and prevents transcription of genes required for cell
proliferation.
Other tumor suppressor/recessive oncogenes involve transcription, cell adhesion and DNA
repair. (see Table 31.1 in book).
Many tumor types appear to require multiple independent mutated oncogenes to progress to
malignancy.
Mutations in ras and p53 are common to many tumor types.
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p53 can suppress growth of transformed cells that have deletions in p53. Thus p53 is a
tumor suppressor.
In some cases, aberrant p53 molecules inhibit the function of normal p53 molecules. Called a
dominant negative mutation, because loss-of-function mutation behaves as if it is an inhibitory
gain-of-function mutation.
p53 forms multisubunit DNA binding protein and one altered subunit could result in loss of
function.
p53 “surveys” for DNA damage. When found it relays that to cell-cycle machinery to stop in G1
until repairs are made. p53 can induce damaged cells to die via apoptosis.
Altered p53 allows cells to replicate damaged DNA giving mutations or gross chromosomal
rearrangements.
P53 mutations may be so prevalent because alterations of many of its amino acids (over 200) has
been shown to be deleterious. These mutations are in the DNA binding domain.
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31. The crystalline lens
Ovoid shaped tissue that is transparent and flexible. Two functions 1) add refractive power and
2) change shape to change the total refractive power.
The lens is made up of anterior surface epithelial cells that undergo mitosis and migrate towards
the equator where they give rise to elongated fiber cells.
Lens Proteins
Crystallins account for 90% of all soluble lens proteins. There are three major types: α (10%),
β (55%) and γ (35%). Major classification of crystallins is based on their relative mobility in an
electric field.
Location
Only α-crystallin is found in the lens epithelial cells. Thus β- and γ-Crystallins are characteristic
of fiber cells.
Not unique to the Eye
It had been thought that crystallins were unique to the eye. However, it has been shown that α-
crystallins are related to heat shock proteins and may have a similar protective function in the
eye. β-crystallins are duplications of standard housekeeping enzymes.
Role in the Lens
Though crystallins are very water soluble, their role in lens function is primarily structural.
They serve to maintain the elongated shape of the lens fibers. All of the crystallins form globular
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polypeptides whose quaternary structure is a multimeric aggregate. Protein content of lens fiber
cells are much greater than that of a typical cell found in the body.
α-crystallins have a chaperone function in that they prevent structural denaturation of all lens
proteins. A mutation in α-crystallin gene could cause a congenital cataract.
Post-translation Modification
Includes acetylation, phosphorylation, glycosylation. Acetylation (the addition of acetyl groups
at the N-terminus) is the most important. It prevents cellular degradation of the protein.
Therefore, crystallins are quite stable (need to last many decades).
Affects of Aging
New lens fibers are continually created. This leads to compaction of the lens with the inner
fibers becoming more dense (less water) and so the refractive index will increase as well.
Cataracts are lens opacities that lead to reduced functional visual acuity. Cataractous lenses have
a higher proportion of insoluble protein than non-cataractous lenses. This is believed to occur
primarily through the formation of disulphide bridges, which produce very high molecular
weight aggregates.
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Current theory is that oxidizing substances (oxygen free radical, H2O2, etc.) will oxidize –SH
groups leading to –S-S- formation.
+
2H2O + 2O2 → 3O2- + 4 H → H2O2 → 2H2O + O2
light SOD catalase
H2O2 oxidizes proteins and lipids. Catalase activity seems to be important since its inhibition
causes cataracts in rabbits.
Arachadonic acid, a component of the lipid membrane, when oxidized can lead to formation of
lipid peroxides: prostaglandin G, thromboxane B2 and 5 -Hpete. These compounds can alter
membrane permeability (mechanism?) and they are produced by activated immune cells.
Increased membrane permeability reduces the effectiveness of ion pumps needed to maintain
osmotic balance.
Protective Mechanisms
-Superoxide Dismutase (SOD)
- catalase
- glutathione peroxidase
2G-SH + H2O2 → G-S-S-G + H2O
-glutathione reductase
G-S-S-G + NADPH + H+ → 2G-SH + NADP+
NADPH comes from the pentose phosphate pathway, which is very active in the lens.
- aqueous has high [ascorbate], which is a general antioxidant
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Schematic diagram shows that large aggregate formation can disrupt membranes. Membrane
disruption will cause leakage of ions, water and protein. End result is water influx. Light scatter
occurs because the uniform density of refractive index has been disrupted since there are
aggregates (high index) and watery areas in close proximity to each other.
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32. COLLAGEN
Collagen is a major structural protein in the body and of great importance to the eye. Collagen is
produced by fibroblasts. 80-90% of the eye is collagen. Collagen is extracellular and insoluble.
Collagen provides the toughness and structure of the outer tunic of the eye (Sclera and cornea).
Collagen is also a major component of the vitreous gel. The basic unit of collagen is a three
stranded triple helix protein called tropocollagen. Each strand is a helix (minor helix) and the
three chains together form a helical structure (major helix). There are at least 8 types of collagen
(see chart). Common features are hydroxylated proline and lysine (require Vitamin C cofactor).
The major type of collagen in the eye is type I.
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33. Photoreceptor Biochemistry
The photoreceptor layer of the retina is the outermost layer of the neural retina. It
is made up of densely packed rods and cones.
1. Rod photoreceptor cells contain the photopigment rhodopsin.
a. Seven loop transmembrane protein- opsin
b. 11-cis retinal chromophore linked to opsin is the light sensitive molecule
(rhodopsin has an absorption peak at 500 nm.)
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c. the sequence of the cone opsins determines the wavelength of light that
activates the chromophore (11 cis-retinal).
c1. 11-cis retinal associates with three cone opsins (blue, green and red).
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c2. Genes for rhodopsin are on chromosome 3 and the gene for blue
opsin is on chromosome 7. Green and red opsins are on the X
chromosome. Thus mutations that lead to inactive or altered pigments
are more prevalent in males.
c3. –OH containing amino acids (serine and threonine) are responsible
for most of the differences between red and green photopigments.
C4. Point mutations in these residues shifts the absorption maximum 10
nm to the red.
C5. Polymorphism at residue 180 (62% Ser, 38% Ala) of the X-linked
red visual pigment apoprotein is responsible for differences in color
matching among males with normal color vision.
2. The photopigment can freely diffuse in the plasma membrane and the
membrane discs. Structural mutations in rhodopsin or associated proteins can
cause photoreceptor degeneration. Called retinitis pigmentosa.
3. Disc Renewal is active and rapid.
a. pulse label moves distally and is shed into RPE cells.
b. entire set of discs is replaced about every 10 days (accelerated in the dark).
c. phagocytosis of disc segments is orchestrated by the RPE, which then
degrades the discs. Incomplete degradation of shed discs leads to an
accumulation of lipofucsin and drusen. Due to aging of the lysosomal
degradation process. Accumulation linked to macular degeneration.
d. RPE is filled with melanin to absorb stray light.
4. The chromophore: (this is what actually adsobs the light). Light stimulation of
rhodopsin results in isomerization of 11-cis retinal to all trans-retinal.
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5.
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5. Retinoids are hydrophobic and therefore need blood carrier proteins.
a. retinols are stored in liver and in blood are bound to serum retinol binding
protein (SRBP) and transthyretin.
b. Receptors for this complex are present on the basolateral surface of RPE
cells.
c. On bonding the retinoid diffuses into the RPE and the carriers are released.
d. Retinoid binds cellular retinol (associating protein) binding protein (CRBP).
e. Trans retinol is converted to 11-cis retinal in RPE cells and is now bound to
cellular retinaldehyde binding protein (CRALBP).
f. At the apical surface of the RPE 11-cis retinal is transferred to
interphotoreceptor binding protein (IRBP).
g. IRBP then delivers it to the photoreceptors where it binds opsin.
h. Light absorption causes conversion from 11-cis to all-trans retinal, which is
then reduced to retinol and released from opsin.
i. All-trans retinol is oxidized and isomerized to 11-cis retinal in the RPE.
Therefore, some recycling occurs. Degraded discs not recycled.
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Primary precursor to vitamin A is β-carotene from plant products. Additional
source is retinyl esters from animal products.
Retinol can act as a developmental hormone (it modifies protein synthesis).
Another form of vitamin A, retinoic acid, supports production of glycoproteins
and epithelial cell differentiation. Lack of retinoic acid leads to degeneration of
the corneal epithelium. Thus deficiency of vitamin A can lead to decreased
night vision (nyctalopia) and a hardening or degeneration of the conjunctiva and
corneal epithelium with loss of mucous and fluid secretions (Xerophthalmia).
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6. PHOTOTRANSDUCTION
a. a photon of light is absorbed by rhodopsin chromophore, 11-cis retinal
causing isomerization to all-trans retinal.
b. Activated rhodopsin decays through several intermediates in approx. 1 ms to
form metarhodopsin II, which is the R* activated species. R* can last for
about one minute.
In Photoreceptor
In RPE
c. R* can then bind to transducin, a trimeric G-protein (Tαβγ) causing exchange
of GDP for GTP.
d. GTP binding causes dissociation of α subunit.
e. Tα binds to a phosphodiesterase (PDEαβγ), releasing the gamma inhibitory
subunit, which binds to Tα, thereby activating PDE.
f. Activated PDE hydrolyses cGMP
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g. cGMP normally keeps a plasma membrane cation channel open. So,
hydrolysis of cGMP acts to close the channel. This hyperpolarizes the
membrane potential.
(-)
Recoverin
Ca2+
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7. Recovery
a. Tα-PDE-γ complex hydrolyses its bound GTP and Tα becomes inactive.
b. Tα dissociates from PDEγ and PDEγ reassociates with PDE, which
becomes inactive.
c. Tα reassociates with Tβγ.
d. The concentration of cGMP then increases by the action of guanylate
cyclase that makes cGMP from GTP and thus the cGMP gated channel
reopens.
e. Activated rhodopsin is phosphorylated by rhodopsin kinase.
f. Phospho-rhodopsin is recognized by arrestin, which binds and inactivates it,
so it can’t activate any more transducins.
g. The rhodopsin chromophore separates from the opsin protein. The
chromophore is regenerated in the RPE.
8. AMPLIFICATION
a. a single photon of light activates one rhodopsin molecule
b. one rhodopsin can activate 500 transducins
c. 500 transducins can activate 500 PDE molecules
d. 500 PDE molecules hydrolyze 10,000 cGMP molecules
e. the loss of 500 cGMP molecules closes 250 channels.
+
f. 250 channels lock out 1-10 million Na ions.
g. the photoreceptor cell membrane potential is hyperpolarized by 1 mv
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9. Adaptation (When you are dark adapted very little light is needed for you to say
you see a change in light intensity. However, when you are light adapted, a bigger
change in light input is needed for you to say that you can see a change.)
a. phototransduction cascade must be down regulated in order to function in
bright light.
b. This is done through changes in cGMP and Ca2+ concentrations. These
molecules interact with primary phototransduction proteins or other
regulatory proteins.
10. Adaptation Mechanisms
a. PDEγ has GTPase activity, so when PDEγ is dissociated (more is dissociated
if there is lots of light) it can hydrolyze the GTP of active Transducin to
GDP, thereby inactivating Transducin. This situation will occur as an
adaptation to high amounts of light.
b. Ca2+ binds to a regulatory protein called recoverin that binds to Rhodopsin
kinase and inhibits it, thus R* is preserved. In the light, [Ca2+] goes low so
Rhodopsin kinase is not inhibited and R* is quickly inactivated. On the
other hand, in the dark Ca2+ is high so the lifetime of R* is longer.
c. Ca2+ also interacts with the cGMP gated channel association protein and
decreases its affinity to cGMP (shifts the Km to the right). Ca2+ is high in
the dark, so a small drop in [cGMP] will decrease channel binding and close
channels. In the light, Ca2+ is low so the affinity for cGMP is higher so a
greater stimulus (i.e., greater drop in [cGMP]) is needed to close the
channel.
d. Ca2+ slows the activity of guanylate cyclase. Ca2+ is low in the light,
therefore guanylate cyclase activity is high and lots of cGMP is being made.
If lots of cGMP is around the channel will tend to stay open and so more
light input is needed to close any cGMP gated channels. In the dark, the
opposite is true, so the photoreceptor is more sensitive.
e. At very high light levels (looking at sun reflected from snow or at the beach)
the amount of photoisomerization is limited because of the finite rate at
which the chromophore can be regenerated.
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