Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity.
Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.
Matsuda
Y
Y
Department of Biochemistry, Osaka University Medical School, Japan.
Higashiyama
S
S
Kijima
Y
Y
Suzuki
K
K
Kawano
K
K
Akiyama
M
M
Kawata
S
S
Tarui
S
S
Deutsch
H F
HF
Taniguchi
N
N
D002850:Chromatography, Gel; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds; D013482:Superoxide Dismutase / Q000302:isolation & purification* / Q000378:metabolism
D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D013482:Superoxide Dismutase