The killing of microorganisms by phagocytic leukocytes is a major mechanism of defense against bacterial infection in humans. Upon contact with a microorganism, the phagocytic leukocyte undergoes a """"""""respiratory burst"""""""" that leads to the production of active oxygen species. Essential features of this process are activation of a membrane-bound oxidase that catalyzes the reduction of oxygen to superoxide using NADPH as the principal electron donor, conversion of superoxide to hydrogen peroxide through spontaneous reaction and catalysis by the enzyme superoxide dismutase, and regeneration of NADPH levels through increased activity of the hexose monophosphate shunt. Hypochlorous acid, formed by the action of leukocyte myeloperoxidase on hydrogen peroxide and chloride ion, is an especially effective antimicrobial agent. Although not the only means of bacterial killing, the peroxide-halide-myeloperoxidase system appears to be the most potent one utilizing hydrogen peroxide. The mechanism by which hypochlorite kills bacteria, however, has not been fully elucidated. The present proposal is based on recent evidence indicating the direct chlorination of NADH and NADPH by myeloperoxidase and other hypochlorite-generating peroxidases. Such a reaction is important because it has the potential of immediately and irreversibly inhibiting metabolism. In the proposed studies, the chlorinated pyridine nucleotides will be isolated by chromatography and their structures determined by NMR techniques. Possible inhibitory properties of chlorinated NAD and NADP will be tested on enzymes such as glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehyrogenase. To correlate the killing of E. coli with the chlorination of its pyridine nucleotides, the organism will be grown in the presence of 14-C-nicotinic acid and exposed to hypochlorite-generating conditions. The radioactivity in cell extracts that co-chromatographs with authentic chlorinated NAD and NADP will then be correlated with the degree of cell killing as determined by viable cell counts. Formation of inhibitory pyridine nucleotide analogs has long been known to be the basis of cellular toxicity of compounds such as 6-aminonicotinamide. However, toxicity of the chlorinated pyridine nucleotides would be more acute because of the rapidity of their formation.
| Bernofsky, C (1991) Nucleotide chloramines and neutrophil-mediated cytotoxicity. FASEB J 5:295-300 |
| Bernofsky, C; Bandara, B M; Hinojosa, O (1990) Electron spin resonance studies of the reaction of hypochlorite with 5,5-dimethyl-1-pyrroline-N-oxide. Free Radic Biol Med 8:231-9 |
| Bernofsky, C; Bandara, B M; Hinojosa, O et al. (1990) Hypochlorite-modified adenine nucleotides: structure, spin-trapping, and formation by activated guinea pig polymorphonuclear leukocytes. Free Radic Res Commun 9:303-15 |
| Bernofsky, C; O'Dea, S W (1986) Nucleotide modification, a radical mechanism of oxidative toxicity. Free Radic Res Commun 2:129-36 |
