Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis.
A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.
D000596:Amino Acids / Q000032:analysis; D001441:Bacteroides fragilis / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D002712:Chlorides; D004589:Electrophoresis, Disc; D004790:Enzyme Induction / Q000187:drug effects; D005296:Ferrous Compounds; D007526:Isoelectric Point; D008345:Manganese / Q000032:analysis*; D017895:Manganese Compounds; D010100:Oxygen / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000302:isolation & purification
D000596:Amino Acids; D002712:Chlorides; D005296:Ferrous Compounds; D017895:Manganese Compounds; D000644:Quaternary Ammonium Compounds; D008345:Manganese; C038178:ammonium ferrous sulfate; D013482:Superoxide Dismutase; C025340:manganese chloride; D010100:Oxygen