Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver.
1. A basic protein (pI = 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn-superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25,000. This larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr 2,400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immuno-blotting to be exclusively localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organ-specific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein.
Institut für Physiologische Chemie, Universität Düsseldorf.
T
T
Ishikawa
A R
AR
Hunaiti
G
G
Piechot
B
B
Wolf
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013482:Superoxide Dismutase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D046911:Macromolecular Substances; D013482:Superoxide Dismutase