Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas.
Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60 degrees C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD.
Osatomi
K
K
Graduate School of Science and Technology, Nagasaki University, Bunkyo, Nagasaki, 852-8521, Nagasaki, Japan. osatomi@net.nagasaki-u.ac.jp
Masuda
Y
Y
Hara
K
K
Ishihara
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003300:Copper; D004064:Digestive System / Q000737:chemistry / Q000201:enzymology*; D005399:Fishes; D005432:Flounder / Q000378:metabolism*; D007525:Isoelectric Focusing; D007564:Japan; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D015032:Zinc
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc