Characterization of superoxide dismutase from Actinomyces
Barkley, Katherine Byer
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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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