This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. The main studies performed as part of this research effort during the past year are summarized below: Project 1. We previously has been demonstrated that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) results in male infertility due to defects in sperm function that include a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD+ cofactor essential for activity of GAPDHS, or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance (NMR) analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD/NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the LDH inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported a previous proposal that lactate dehydrogenase A, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC. By co-immunoprecipitation coupled with mass spectrometry we have identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport and/or sequestration. On the basis of these results, it appears that LDHC is part of a complex involved in ATP homeostasis that is disrupted in spermatozoa lacking this isozyme. Project 2. Copper-zinc superoxide dismutase (SOD) protects the cell as a consequence of its role in the dismutation of superoxide. However, in the presence of hydrogen peroxide it can also generate a powerful oxidant, the carbonate anion radical, which can in turn oxidize other cellular targets. The mechanism of carbonate radical formation by SOD has been a subject of intense debate. Recent kinetic studies have been designed to provide insight into this process, focusing on the possible role of peroxycarbonate as a substrate for reduced SOD. Based on recent theoretical analyses of SOD, it appears that reduction of the active site Cu(II) to Cu(I) by hydrogen peroxide Is accompanied by release of the bridging histidine ligand to the copper. This may allow larger ions, such as peroxycarbonate, to bind to the Cu(I) enzyme, and the subsequent chemistry might result in formation of the carbonate radical. We have also continued our studies using NMR to evaluate the role of carbonic anhydrase in the metabolism of peroxymonocarbonate. Project 3. We previously used NMR to characterize ligand interactions with a putative acetylcholine binding protein (AChBP) derived from the marine polychaete Capitalla teleta. We have recently begun a related project to investigate ligand binding with an AChBP derived from the black widow spider. The NMR approach that we have followed utilizes competitive binding interactions to allow the determination of dissociation constants much lower than would be feasible using just the ligand and binding protein. These studies begin with a weak binding ligand, such as choline, and subsequently evaluate ligands with decreasing KD values. The spider toxin contains an additional 35 residue extension at the C-terminus, the function of which is unknown.
