Blair by 33GRVMI

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									    dNMP Kinase Activity in
   Mitochondria and Its Role in
   Mitochondrial Mutagenesis

   Brian M. Blair
  Dr. Christopher K.
        Mathews

    Department of
    Biochemistry and
       Biophysics

    Oregon State
      University
 HHMI Research 2007

                        http://www.nsf.gov/news/overviews/biology/assets/interact08.jpg
                                            Why Is Research on mtDNA
                                             Metabolism Important?

                                                                       Semiautonomous
                                                                      mtDNA has 10-100
                                                                         eukaryotic organelle
                                                                       fold higher mutation
                                                                       Responsible for ATP
                                                                       rate than nuclear
                                                                         synthesis
                                                                       DNA
                                                                       Functions linked
                                                                      Mutations passed to:
                                                                           generation
                                                                       fromApoptosis to
                                                                       generationprocess
                                                                           Aging
                                                                      LessSensitivity to anti-
                                                                           effective
                                                                            HIV drugs
                                                                       mtDNA repair
                                                                       Contain their own
                                                                       mechanism
http://en.wikipedia.org/wiki/Intermembrane_space_of_mitochondria
                                                                         genome:
                                                                           mtDNA
                                                                      mtDNA mutations
 http://www.ccc.columbia.edu/Mitochondrial_Diseases/mito/round.gif     = disease
                           Mitochondrial Disease

 Researchers have now                     Diseases
  discovered over 40                      involving altered
  types of mitochondrial                  mitochondrial
  disease                                 function:
                                              Parkinson’s
 40,000-70,000
                                              Disease
  Americans affected
                                              Alzheimer’s
 Many age-related                            Disease
  diseases involve                            Type 2 Diabetes
  defects of
                                              Various Cancers
  mitochondrion
                                              Neurodegenerative
                                              Disorders
                                              Cardiomyopathies
                Deoxyribonucleoside Triphosphates
                            (dNTPs)

 Four mtDNA
  precursors:
    dATP
    dGTP
    dCTP               Deoxyadenosine   Deoxyguanosine
                         triphosphate     triphosphate
    dTTP


 dNTP pool
  asymmetries =
  mtDNA mutagenesis
                        Deoxythymidine    Deoxycytidine
                         triphosphate      triphosphate
   Metabolic Routes to
Intramitochondrial dNTPs

                   Pathways
                    involving
                    dNMPs
                   Formation by
                    salvage route
                   Are the
                    pathways
                    involving
                    dNMPs
                    significant in
                    forming the
                    dNTP pool
                    asymmetries?
                         Purpose
    Design an assay to measure dNMP phosphorylation to
     dNDP within the mitochondrion
    Measure enzymatic activity of dNMP kinase
     Brain, Liver, Heart, Skeletal Muscle, and Kidneys
    3) Measure the dNMP kinase activity using dTMP,
     dGMP, dCMP, and dAMP as substrates



                     Hypothesis #1
 The dNMP kinase activity will vary within the different
  mammalian tissue mitochondria.
 Analysis of this activity will help explain the different
  uptake pathways in dNMP metabolism and possible
  reasons behind dNTP asymmetry.
          dNMP Kinase
dNMP                    dN-P-P*
                         dNDP


       A-P-P-P*
         ATP      ADP
          Method 1: TLC Assay

 Develop assay using T4 infected E. coli
  HB101/pBK5 recombinant
 ATP-γ-P33 to trace activity of dNMP kinase

 Rxn Mixture:
    0.2 M Tris-HCl, pH 7.8; 0.02 M MgCl2; 0.02 M
     ATP; 2.0 mM dTMP
    Use 50 µL rxn mixture with substrate, 0.1 µCi ATP-
     γ-P33, 10 µL rat mitochondrial extract, water to 100
     µL

 Run on TLC in 0.5 M LiCl and 5% Na2B4O7,
  pH 7.0 aqueous solvent
 Measure cpm of dNDP and calculate specific
  activity of enzyme
                                       Results of TLC Data
                      No substrate control consistently has more counts
                       than the with dTMP substrate

        1) Test ATP-γ-P33 for contamination                              2) Original problem still present
                     Assay Test                                                    TLC Reaction w/ New P33

      9000                                                       25000
                                         No Enz/Sub
      8000                                                                            60 min
                                         heat 2 min                                                         0 sub
      7000                                                       20000
                                         P33
      6000
                                                                 15000




                                                           CPM
      5000                                                                30 min                                    dTDP CPM
CPM




      4000                                                       10000                             0 min
      3000
                                                                 5000
      2000
      1000                                                          0
          0                                                                 1           2               3     4
      -1000 0    5      10        15      20          25
                                                                                            Reactions
                        Length (cm)




   Other attempts to fix:                                        Results:
      Remove small molecules or pre-                                Still co-migration occurs
        existing substrates from extract
                                                                     TLC assay cannot be used
      Run TLC in 10 different solvent
        systems
        Method 2: HPLC Assay

 0.2 M Tris-HCl, pH 7.8; 0.02 M MgCl2; 0.02 M
  ATP; 2.0 mM dTMP
    Use 50 µL rxn mixture with substrate, 10 µL rat
     mitochondrial extract, water to 100 µL
    Dilute 20-fold and run in HPLC


 Run standards to label nucleotides during rxn
Nucleotide Standards            ADP



                                        ATP




                                      dTTP
                         dTDP


                   AMP
               dTMP
0’ Rxn                   ATP                    30’ Rxn                       ATP



                                                                      ADP

                                                            1? 2?
     dTMP          ADP                                Thy dTMP




60’ Rxn                  ATP                                1?              ATP     120’ Rxn
                                                                      ADP

         1?        ADP
                                                      Thy        2?
              2?
   Thy
         dTMP                                               dTMP




                          0 dTMP Substrate      ATP


                                1?        ADP


                                     2?
                                      Results of Reactions
                                  dTMP Concentrations vs. Reaction Times

                        3000000




   Area Concentration
                        2500000
                        2000000
                                                             dTMP Concentrations
                        1500000                              No Substrate (dTMP) Control
                        1000000
                        500000
                             0
                                  0         50         100          150
                                            Time (minutes)




                                       Hypothesis #2
 dTMP is getting broken down to thymidine
  and Pi as part of the 5’-deoxynucleotidase
  activity and regulation pathway
             Thy        1mM Thymidine




                         Thy
      dTMP   0 ATP 0’                   0 ATP 60’


Thy
Explanation of dTMP Data

            Removal of ATP
             blocks formation of
             dTMP from
             thymidine

            Result:
               All dTMP gets
                converted to
                thymidine and Pi
               Activity of 5’-
                deoxynucleotidase
                high in rat liver
                mitochondria
                     Summary

Developed assay using thin layer chromatography to measure
   enzymatic activity of dNMP kinase



Co-migration of unknown products at dTDP designated area




Used HPLC to visualize elution times and peak areas; I found
   two unknown reaction products formed



Proved activity of 5’-deoxynucleotidase forming thymidine
   from dTMP in absence of ATP; activity is high in rat liver
   mitochondria
        Future Research Goals

 Continue to perfect the HPLC assay
 Pursue the other reactions that are occurring
  and find the reason behind this occurrence
    Use mass spectrometer to determine molecular
     weight of unknown products
 Measure the dNMP kinase activity of the
  four different deoxynucleotides within the
  different tissue mitochondria
               Acknowledgements

 Howard Hughes Medical
   Institute
 Dr. Christopher K.
   Mathews
 Linda Benson
 Dr. Kevin Ahern
 Oregon State University


 Funding:
     Howard Hughes
      Medical Institute

								
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