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. Lactate dehydrogenase C (LDHC) and energy metabolism in mouse sperm. Disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) results in defects in sperm function, including a more rapid decline in ATP levels over time in sperm from Ldhc-/- (LDHC-KO) mice than in sperm from wild type (WT) mice, a concurrent decrease in progressive motility, and the failure to develop a hyperactivated motility pattern. During the past year, we utilized NMR as well as other methods for determining metabolite concentrations in order to understand the metabolic basis for the above changes in function of sperm derived from Ldhc-/- mice. We found that glucose consumption, monitored with 1-13C-D-glucose, was disrupted in the absence of LDHC. However, no significant differences in the NAD/NADH ratio or pyruvate levels were detected between WT and KO sperm. Surprisingly, we also observed that pyruvate was rapidly metabolized to lactate not only by WT sperm, but also by KO sperm. Moreover, we found that the metabolic disorders induced by treatment with an LDH inhibitor (sodium oxamate) were different from those induced by the lack of LDHC. These results suggest that LDHA, an LDH isozyme associated with the fibrous sheath, is responsible for some or most of the LDH activity in Ldhc-/- sperm, while LDHC has an additional role in the maintenance of energy metabolism in sperm. Co-immunoprecipitation studies demonstrated that LDHC interacts with sperm-specific ADP/ATP transporter 4 (ANT4). This suggests LDHC is associated with a protein complex involved in redistributing ATP generated by glycolysis in sperm. Project 2. We recently have identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic Ach receptor (nAChR) R subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and R-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (KD = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca. Project 3: Richardson and coworkers have recently shown that the oxidative chemistry of hydrogen peroxide can be potentiated in the presence of CO2 as a result of the non-enzymatic formation of peroxymonocarbonate. This species is capable of oxidizing biologically important substrates such as methionine, and limits the availability of peroxide to catalase degradation. However, the biological significance of peroxycarbonate is dependent on the kinetics of its formation as well as its reactivity, and it has been suggested that this may be too slow to be physiologically significant. We have continued to investigate the kinetics of the bicarbonate/peroxycarbonate/carbon dioxide equilibria using 1D and 2D NMR exchange spectroscopy (EXSY), and have also evaluated the effect of carbonic anhydrase on this reaction. In the absence of enzyme, the kinetics of the CO2←→HCO3- reaction are too slow to produce observable cross peaks, however the exchange of CO2 and HCO4- is readily observed. Addition of carbonic anhydrase produces the expected enhancement of carbon dioxide-bicarbonate exchange, but failed to significantly increase the reaction of peroxide with carbon dioxide. However, as a result of the indirect, carbon dioxide-mediated exchange, the exchange of bicarbonate and peroxycarbonate is dramatically increased. We continue to quantify these effects in order to clarify the underlying chemistry of these processes so that we can understand their biochemical significance.
