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McKee 2010 Abstract IOC60

From Bioblast
McKee E, Thomas C, McAbee K, Verwilst L, Thayer T, Chhoy P (2010) Fishing in the mitochondrial deoxynucleotide pools. MiPNet15.10.

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McKee E, Thomas C, McAbee K, Verwilst L, Thayer T, Chhoy P (2010)

Event: IOC60

Introduction: It is well established that an appropriately balanced deoxynucleotide (dNTPs) pools are required for normal mitochondrial DNA replication. Disorders in dNTP metabolism that affect these pools lead to mitochondria DNA depletion diseases which have devastating consequences. However, little is known concerning the mechanisms by which these mitochondrial pools are regulated. In earlier work we demonstrated that isolated intact mitochondria were capable of synthesizing all four deoxynucleotide triphosphates (dNTPs) from radiolabeled deoxynucleoside precursors. The aim of this study was to determine the extent that dNTP pools in isolated mitochondria depend on the concentration of deoxynucleosides in the medium and the extent of transport of the mitochondrially synthesized dNTP pools from the matrix to the medium. We are also interested in investigating the extent that anti-viral nucleoside analogs may perturb these systems. In this regard we have already demonstrated that AZT is a competitive inhibitor of thymidine phosphorylation in mitochondria isolated from heart, liver, and brain [1, 2] and that AZT significantly decreases the size of the mitochondrial TTP pool.

Methods: Tightly coupled mitochondria isolated from adult rat heart were incubated at 300C in an appropriate medium with varying concentrations of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and thymidine (dT). At various time-points, the levels of all four dNTPs in the medium and in the mitochondria were measured using a template driven assay as described [3] that limits the interference with rNTPs.

Results: The results demonstrated that the initial level of the dNTP pools and their response to medium concentration of deoxynucleosides varied considerably with dGTP being the predominant dNTP. TTP was the only dNTP shown to be absolutely dependent on the concentration of its precursor, thymidine in the medium and was also the only mitochondrially synthesized nucleotide to freely equilibrate with the medium. The other mitochondrially synthesized dNTPs appear to be primarily sequestered within the matrix.

Conclusions: These results suggest that the mitochondrial TTP pool is likely to be much more sensitive to perturbation in precursor thymidine levels than the other mitochondrial dNTPs.


[1] McKee EE, Bentley AT, Hatch M, Gingerich J, Susan-Resiga D (2004) Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity. Cardiovasc. Toxicol. 4: 155-167.

[2] Lynx MD, McKee EE (2006) 3'-Azido-3'-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria. Biochem. Pharmacol. 72: 239-243.

[3] Ferraro P, Franzolin E, Pontarin G, Reichard P, Bianchi V (2010) Quantitation of cellular deoxynucleoside triphosphates. Nucleic Acids Res. 38: e85.

β€’ Keywords: deoxynucleosides; deoxynucleotides


Labels:

Stress:Mitochondrial Disease; Degenerative Disease and Defect"Mitochondrial Disease; Degenerative Disease and Defect" is not in the list (Cell death, Cryopreservation, Ischemia-reperfusion, Permeability transition, Oxidative stress;RONS, Temperature, Hypoxia, Mitochondrial disease) of allowed values for the "Stress" property. 

Tissue;cell: Heart  Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Mitochondrial Biogenesis; Mitochondrial Density"Mitochondrial Biogenesis; Mitochondrial Density" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 


Event: IOC60"IOC60" is not in the list (A1, A2, A3, A4, B1, B2, B3, B4, C1, C2, ...) of allowed values for the "Event" property.