Heart Mitochondrial TTP Synthesis and the Compartmentalization of TMP [Enzymology]

December 11th, 2014 by Kamath, V. G., Hsiung, C.-H., Lizenby, Z. J., McKee, E. E.

The primary pathway of TTP synthesis in the heart requires thymidine salvage by the mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [3H]-thymidine or [3H]-TMP as precursors of [3H]-TTP in isolated intact or broken mitochondria from rat heart. The results demonstrated that [3H]-thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP. The equivalent addition of [3H]-TMP produced far less [3H]-TTP than the amount observed with [3H]-thymidine as the precursor. Using AZT to inhibit TK2 the synthesis of [3H]-TTP from [3H]-TMP was effectively blocked, demonstrating that synthesis of [3H]-TTP from [3H]-TMP arose solely from the de-phosphorylation of [3H]-TMP to [3H]-thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [3H]-thymidine was readily converted to [3H]-TMP but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain as confirmed by addition of FCCP and KCN, respectively in intact mitochondria. In summary, these data taken together suggest that the thymidine salvage pathway is compartmentalized such that TMP kinase prefers TMP synthesized by TK2 over medium TMP, and this is disrupted in broken mitochondria.