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Browsing by Author "Moore, L.V.H."

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    Characterization of superoxide dismutase from Actinomyces
    Barkley, Katherine Byer (Virginia Polytechnic Institute and State University, 1988)
    The anaerobes Actinomyces naeslundii, A. odontolyticus and Actinomyces strain ii E1S.25D produce a Mn-containing superoxide dismutase (MnSOD). Actinomyces, once classified as yeast based on their morphology, are saprophytic organisms found among the normal flora of the mouth but can act as endogenous pathogens resulting in gingivitis and actinomycosis. The ability of Actinomyces to scavenge superoxide may increase survival of the cell from the O₂⁻-dependent killing by polymorphonuclear leukocytes and also enable the organism to be transported through an oxygenated environment from one site to another. The MnSODs were purified 85-240 fold from crude extracts with 30-60% yield by two chemical fractionations and three chromatography steps. The enzymes, Mr 96,000, were tetramers of equally sized, noncovalently associated subunits similar to the MnSOD found in Saccharomyces cerevisiae. Each of the Actinomyces MnSODs contained 0.5 g-atoms Mn/subunit and were stable in the presence of 1 mM NaCN, 1 mM NaN₃ and 2.5 mM H₂O₂. The MnSODs from Actinomyces have isoelectric points of 4.2-4.6 and are negatively charged at physiological pH. Amino acid analyses of the high molecular weight MnSODs from Actinomyces, yeast, chicken liver, and Thermus thermophilus indicated similar composition of each subunit. The second order rate constants of each Actinomyces MnSOD were measured at pH 7.8 and found to be in the range of 0.9 - 2.8 x 10⁹ M⁻¹ sec⁻¹ as compared to the rate of 1.8 x 10⁹ M⁻¹ sec⁻¹ for yeast MnSODs. Structural relatedness was evaluated by immunological studies. Rabbit antisera to each of the Actinomyces MnSODs were prepared. The MnSODs from A. naeslundii and Actinomyces strain E1S.25D both showed complete identity with their respective antibodies and partial identity with the antibody prepared against A. odontolyticus MnSOD. None of the antisera cross reacted with bovine Cu/Zn SOD, Bacteroides Fe- or MnSOD or MnSODs from either Haemophilus influenzae, Deinococcus radiodurans, or S. cerevisiae.
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    The role of molybdenum in the formate dehydrogenase of methanobacterium formicicum
    May, Harold Douglas (Virginia Polytechnic Institute and State University, 1987)
    An examination of oxidation products of the pterin cofactor in the formate dehydrogenase of Methanobacterium formicicum revealed that the cofactor is a 6-substituted pterin similar to the molybdopterin of xanthine oxidase. In contrast to the molybdopterin from xanthine oxidase the formate dehydrogenase cofactor was unable to complement the cofactor-deficient nitrate reductase of Neurospora crassa mutant nit-1 and possessed two phosphate groups. Incubation of oxidized formate dehydrogenase with cyanide resulted in an irreversible loss of enzyme activity which could not be restored by treatment with sulfide. Equimolar amounts of thiocyanate were released from cyanide-treated formate dehydrogenase suggesting the loss of one terminal sulfur ligand to molybdenum. These results along with electron paramagnetic resonance spectroscopy of the cyanide-inactivated formate dehydrogenase suggest that the the molybdenum ligands of the enzyme are similar to those of xanthine oxidase. The concentration of molybdenum and the level of formate dehydrogenase activity in extracts of hydrogen-grown Methanobacterium formicicum decreased by at least 10- fold when the organism was grown for several transfers in molybdenum-deficient media or with added tungstate. Immunochemical analysis showed that both subunits of the formate dehydrogenase were produced regardless of the growth condition. However the amount of formate dehydrogenase protein decreased more than 10-fold when the amount of molybdenum in the cell was low. The pterin cofactor was present in the inactive enzyme from tungstategrown cells; however the protein contained less than 0.05 molecules of molybdenum or tungsten per formate dehydrogenase. Messenger RNA specific for fdh gene was detected in high amount in cells grown without added molybdenum and in low amount in cells that contained high amounts of molybdenum. These results suggest that molybdenum is required for the synthesis of a stable formate dehydrogenase and that a molybdenum-dependent repressor may be required for the termination of fdh transcription.