Transcript dNMP Kinase Activity in Mitochondria and Its Role in Mitochondrial Mutagenesis  Brian M.

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?
http://en.wikipedia.org/wiki/Intermembrane_space_of_mitochondria
http://www.ccc.columbia.edu/Mitochondrial_Diseases/mito/round.gif
Semiautonomous
mtDNA
has 10-100
eukaryotic
organelle
fold
higher mutation
rate
Responsible
for ATP
than nuclear
synthesis
DNA
Functionspassed
linked to:
Mutations
from
generation to
 Apoptosis
generation
 Aging process
 Sensitivity
 Less
effectiveto antiHIV repair
drugs
mtDNA
mechanism
Contain their own
genome:
 mtDNA
 mtDNA
mutations =
disease
Mitochondrial Disease
 Researchers have now
discovered over 40
types of mitochondrial
disease
 40,000-70,000
Americans affected
 Many age-related
diseases involve
defects of
mitochondrion
 Diseases
involving altered
mitochondrial
function:
Parkinson’s
Disease
Alzheimer’s
Disease
Type 2 Diabetes
Various Cancers
Neurodegenerative
Disorders
Cardiomyopathies
Deoxyribonucleoside Triphosphates
(dNTPs)
 Four mtDNA
precursors:
 dATP
 dGTP
 dCTP
 dTTP
Deoxyadenosine
triphosphate
Deoxyguanosine
triphosphate
Deoxythymidine
triphosphate
Deoxycytidine
 dNTP pool
asymmetries =
mtDNA mutagenesis
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
-P-P*
dNDP
dN
dNMP
ATP
A-P-P-P*
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 ATPγ-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
7000
60 min
5000
4000
3000
15000
5000
1000
0
0
5
10
15
20
Length (cm)
 Other attempts to fix:
 Remove small molecules or preexisting substrates from extract
 Run TLC in 10 different solvent
systems
25
30 min
dTDP CPM
10000
2000
-1000 0
0 sub
20000
CPM
6000
CPM
25000
No Enz/Sub
heat 2 min
P33
8000
0 min
1
2
3
4
Reactions
 Results:
 Still co-migration occurs
 TLC assay cannot be used
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
dTDP
AMP
dTMP
dTTP
0’ Rxn
30’ Rxn
ATP
ATP
ADP
1? 2?
dTMP
ADP
60’ Rxn
Thy dTMP
1?
ATP
ATP
ADP
1?
Thy
ADP
Thy
2?
dTMP
2?
dTMP
0 dTMP Substrate
1?
ADP
2?
ATP
120’ Rxn
Results of Reactions
dTMP Concentrations vs. Reaction Times
Area Concentration
3000000
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
dTMP
Thy
0 ATP 0’
1mM Thymidine
Thy
0 ATP 60’
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