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: 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 nono-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. During the past year, we investigated 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. 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 significant 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. Current experiments are addressed and quantifying these exchange effects and clarifying the underlying chemistry of these processes. Project 2. This project is a collaborative study with the Mason group (LP) of the peroxidase activity of superoxide dismutase and the role which peroxycarbonate radical might play in this biochemistry. Although the principle catalytic activity of the enzyme superoxide dismutase (SOD) is believed to be the scavenging and detoxification of superoxide radicals, there is substantial evidence demonstrating that the enzyme may also act as a peroxidase. Further, this activity has been implicated in the onset and progression of familial amyotrophic lateral sclerosis (FALS). We have evaluated the hypothesis that peroxymonocarbonate may play a significant role in the oxidation of the Cu(I), to produce Cu(II), and thereby contribute to the redox cycling of the enzyme. The carbonate radical anion would be produced as a byproduct of this reaction. NMR spectroscopy and electron spin resonance studies support the conclusion that peroxycarbonate is an intermediate in the SOD peroxidase cycle, and serves as a precursor of carbonate radical anions. Project 3. The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between lactate and pyruvate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. Disruption of ldhc severely impairs fertility in male ldhc-/- mice. We have initiated studies of the metabolism of 1-13C-D-glucose in spermatozoa derived from LDHC-KO mice. Glucose uptake/metabolism was severely impaired in spermatozoa derived from the ldhc -/- mice, although they were able to utilize pyruvate. Several other labeled metabolites have been identified, including glycerol and acetate. We are currently studying extracts derived from these cells in order to further characterize the metabolic perturbation, and ultimately the basis for impaired fertility.

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Wang, Huanchen; DeRose, Eugene F; London, Robert E et al. (2014) IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family. Nat Commun 5:4178
Leinisch, Fabian; Jiang, Jinjie; DeRose, Eugene F et al. (2013) Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism. Free Radic Biol Med 65:1497-1505
Odet, Fanny; Gabel, Scott; London, Robert E et al. (2013) Glycolysis and mitochondrial respiration in mouse LDHC-null sperm. Biol Reprod 88:95
Ranguelova, Kalina; Ganini, Douglas; Bonini, Marcelo G et al. (2012) Kinetics of the oxidation of reduced Cu,Zn-superoxide dismutase by peroxymonocarbonate. Free Radic Biol Med 53:589-94
McCormack, Thomas; Petrovich, Robert M; Mercier, Kelly A et al. (2010) Identification and functional characterization of a novel acetylcholine-binding protein from the marine annelid Capitella teleta. Biochemistry 49:2279-87
Bonini, Marcelo G; Gabel, Scott A; Ranguelova, Kalina et al. (2009) Direct magnetic resonance evidence for peroxymonocarbonate involvement in the cu,zn-superoxide dismutase peroxidase catalytic cycle. J Biol Chem 284:14618-27
Gabel, Scott A; London, Robert E (2008) Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry. J Biol Inorg Chem 13:207-17