Superoxide dismutase from Thermus aquaticus. Isolation and characterisation of manganese and apo enzymes.
Superoxide dismutase has been isolated and characterised from the extreme thermophile Thermus aquaticus. The pure enzyme is a reddish-purple manganese-containing protein with a molecular weight of approximately 80000 +/- 5000. Combination of gel electrophoresis in dodecylsulphate and amino acid analysis shows that it is composed of four identical subunit polypeptide chains consisting of approximately 186 amino acids. The tetrameric protein contains two atoms of manganese. A stable manganese-free apoprotein has been prepared by treatment with EDTA in 8 M urea at acidic pH. The apoprotein regains the tetrameric structure in the absence of manganese but is inactive. Reconstitution of active Mn-enzyme was achieved byaddition of Mm2+ apoprotein in 8 M urea at acid pH. Reconstitution was monitored by absorption spectroscopy, manganese analysis and regain of activity and by these criteria the reconstituted enyzme with two atoms Mn per mole is indistinguishable from the native enzyme. The enhanced stability of the thermophile apoenzyme and Mn-enzyme is of advantage for studies of the structure and mechanism of action of superoxide dismutase. The N-terminal amino acid sequence to the 40th residue of the submit was determined by automated Edman degradation. The sequence has a close resemblance to that of the dimeric Mn-enzyme from another thermophile, Bacillus stearothermophilus.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D004355:Drug Stability; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013045:Species Specificity; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D001051:Apoenzymes; D012967:Sodium Dodecyl Sulfate; D008345:Manganese; D013482:Superoxide Dismutase