Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron.
Superoxide dismutase (SOD) from extracts of anaerobically maintained Bacteroides thetaiotaomicron was a dimer of equally sized 23,000-molecular-weight monomers joined noncovalently. A preparation with a specific activity of 1,200 U/mg contained 1.1 g-atom of Fe, 0.6 g-atom of Zn, and less than 0.05 g-atom of Mn per mol of dimer. The apoprotein, prepared by dialysis of iron-SOD in 5 M guanidinium chloride-20 mM 8-hydroxyquinoline, had no superoxide-scavenging activity when renatured without exogenous metal. Enzymatic activity was restored to the denatured apoprotein by dialysis against either 1 mM Fe(NH4)2 or 1 mM MnCl2 in 20 mM Tris (pH 7.0). The Fe-reconstituted enzyme and the native enzyme were inhibited approximately 50% by 0.2 mM NaN3, whereas the Mn-reconstituted enzyme was inhibited 60% by 10 mM NaN3. Aeration of the anaerobic cells resulted in a fourfold induction of an azide-resistant SOD. The enzyme (43,000 molecular weight) isolated from aerated cells was a dimer of equally sized subunits. The metal content was 1.0 g-atom of Mn, 0.55 g-atom of Fe, and 0.3 g-atom of Zn per mol of dimer. Enzymatic activity of the denatured apoprotein from this enzyme was also restored on addition of either iron or manganese. The constitutive Fe-SOD and the O2-induced Mn-SOD, tested alone and in combination, migrated identically on acrylamide gels, had similar amino acid compositions, and had alanine as the sole N-terminal amino acid. These data are consistent with the synthesis of a single apoprotein in either anaerobically maintained or oxygenated cells. We have observed a similar phenomenon with SOD from Bacteroides fragilis (E. M. Gregory, Arch. Biochem. Biophys. 238:83-89, 1985).
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D000596:Amino Acids / Q000032:analysis; D001386:Azides / Q000494:pharmacology; D001439:Bacteroides / Q000201:enzymology*; D007501:Iron / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification*; D008345:Manganese / Q000378:metabolism*; D019810:Sodium Azide; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000302:isolation & purification*; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D001386:Azides; D007527:Isoenzymes; D008345:Manganese; D019810:Sodium Azide; D007501:Iron; D013482:Superoxide Dismutase; D015032:Zinc