ASTI Anatomy and Physiology Example Laboratory Report Steven Fong 9-04-08 Cell Transport Lab Background: Key Terms: Selective Permeability: allowing some solutes/substances to enter the cell while keeping others out. Concentration Gradient: difference in concentration Active Transport: Cell provides energy (often ATP) to power transport Passive Transport: process is driven by concentration or pressure differences Diffusion: movement of molecules from a region of high concentration to low Osmosis: diffusion of water Facilitated Diffusion: solutes that are transported down their gradient by carrier proteins because they are too big or too insoluble MWCO (Molecular Weight Cut Off): Term used to describe the pore size of a membrane Filtration: process where water and solutes pass through a membrane due to pressure Concepts: The larger the MWCO, the larger the pores in the membrane, the more things can pass through. Facilitated diffusion relies on carrier proteins so the rate varies with the number of proteins. If a semipermeable membrane is used, some solutes will be unable to move and water will move instead. If the system is closed and volumes cannot increase, osmotic pressure will be generated. Filtration is not selective. The amount of fluid depends almost entirely on the pressure. Active transport pumps require energy to function as they move against a concentration gradient. Activity 1: Simple Diffusion Purpose: To investigate the effects of membrane pore size on simple diffusion. Hypothesis: If pore size is increased, more solutes will be able to pass through the membrane. Data Table 1: Dialysis Results (average diffusion rate in mM/min) Solute Membrane (MWCO) 20 50 100 200 Na+ 0 0.0150 0.0150 0.0150 Urea 0 0 0 0 Albumin 0 0 0 0 Glucose 0 0 0 0.0040 Notes/Questions: 1. Both Na+ and glucose diffused from the left into the right 2. Urea and Albumin did NOT diffuse 3. To separate urea and albumin we would need membranes with larger MWCOs. 4. To remove urea from a solution that also contained NaCl you would need an appropriate membrane MWCO (big enough for Urea) and a solution in the right beaker with an equal concentration of NaCl so that so net NaCl would leave. This would need to be repeated many times until as much urea was removed as possible. Activity 2: Facilitated Diffusion Purpose: To investigate the affect of varying transport proteins on solute transport rate. Hypothesis: Increasing the number of transport proteins increases the rate of solute transport. Data Table 2: Facilitated Diffusion Results (glucose transport rate, mM/min) Glucose Concentration Number of Glucose Carriers 500 700 900 2.00 0.0008 0.0010 0.0012 8.00 0.0023 0.0031 0.0038 Notes/Questions: 1. As the number of protein carriers increased the rate of facilitated diffusion increased. 2. If you began at equilibrium there would be no net rate of transport. 3. NaCl should have no effect if it can pass through the membrane. Activity 3: Osmosis Purpose: To investigate the effect of solute concentration on osmotic pressure in a closed system. Hypothesis: The greater the concentration gradient in a closed system, the greater the osmotic pressure. Data Table 3: Osmosis Results (pressure in mm Hg) Solute Membrane (MWCO) 20 50 100 200 Na+ 272 0 0 0 Albumin 153 153 153 153 Glucose 170 170 170 0 Notes/Questions: 1. Yes. Osmotic pressure builds in all cases except for Na+ at 50 MWCO and above and Glucose at 200. 2. Yes – all MWCO over 50. 3. An increase in solute concentration leads to an increase in pressure. 4. If solutes are able to diffuse osmotic pressure will NOT be generated as the solute will equilibrate. 5. See answer 4. At equilibrium there will no net movement of water. 6. The pressure would increase. 7. The glucose would equilibrate and osmotic pressure should eventually reach the same level. 8. The glucose would not be able to transport and there would be a higher solute concentration in the RIGHT beaker and thus osmotic pressure would build there. Activity 4: Simulating Filtration Purpose: To investigate the effect of pressure and pore size on the rate of filtration. Hypothesis: The more you increase pressure and/or pore size, the faster the rate of filtration. Data Table 4: Filtration Results Solute Membrane (MWCO) 20 50 100 200 Filtration Rate 1 2.5 5 NaCl In filtrate (mg/mL) 0 4.81 4.81 4.81 Membrane Residue (+/-) + + + + Urea In filtrate (mg/mL) 0 0 4.74 4.74 Membrane Residue (+/-) + + + + Glucose In filtrate (mg/mL) 0 0 0 4.39 Membrane Residue (+/-) + + + + Powdered In filtrate (mg/mL) 0 0 0 0 Charcoal Membrane Residue (+/-) + + + + Notes/Questions: 1. As MWCO increased, filtration rate increased. 2. Powdered charcoal did not appear in any filtrate. 3. Increasing the driving pressure also increases filtration rate. 4. In living membranes you could increase filtration rate by increasing pressure (constriction of vessels) or increasing the size of gaps (pores) within the membrane. 5. The molecular weight of glucose must be greater than 100 but less than 200. Activity 5: Active Transport Purpose: To investigate the effect of ATP on active transport. Hypothesis: Increasing the concentration of ATP will allow more active transport to occur. Notes/Questions: 1. Na+ transport stops before transport has completed because all ATP has been exhausted and the pumps can no longer function. 2. Dispensing no ATP would result in no transport. 3. Increasing ATP increases the amount of Na+ that is transported. 4. Decreasing the number of pumps would reduce the amount of solute transported. 5. If you set the concentration of Na+ to be equal on both sides of the membrane there will still be transport. 6. The lack of K+ means transport cannot occur since the pump only works when it has both Na+ and K+ present. 7. As long as there is sufficient ATP, increasing the number of pump proteins will increase transport. 8. Adding glucose would not increase transport. Conclusion/Discussion: In this lab we investigated the effect of several factors on the rate of transport across membranes. In the first activity we found that as pore size increases, more types of solutes are able to diffuse across the membrane. However, once pore size is large enough, increasing it further does not affect the rate of transport. In the second activity we found that in facilitated diffusion increasing the number of carrier proteins increases the rate of transport as does increasing the concentration of solute. However, it is expected that once solute concentration is high enough and all carriers are allowing transport at their maximum rate no further increase in solute concentration would have an effect. In the osmosis investigation we found that in a closed system, osmotic pressure is generated when there is a solute that cannot pass through the membrane. This occurs because when solute cannot move, water attempts to move across the membrane to balance. Since the system is closed and volume cannot increase, pressure builds. In the model of filtration, it was found that increasing the pore size and/or pressure increases the filtration rate. In the final activity we found that increasing the concentration of ATP provides more energy to protein pumps which results in more overall transport. The amount of active transport is unaffected by the addition of solutes on either side of the membrane as it is not dependent on concentration gradients. All hypotheses were supported in this lab and results were as expected. These results demonstrate several fundamental concepts of membrane transport and help to explain many physiological functions. For example, blood pressure can play a strong role in the rate of filtration in the kidney. This allows the body to maintain proper osmotic balance. In transporting nutrients into the body from the digestive tract, facilitated diffusion and active transport are employed. Cells maintain their osmotic pressure by regulating the solute concentrations in their cells and in some cases solute concentrations can lead to dangerous pressures. This can happen with blood pressure due to disease such as diabetes.