Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability.
A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.
Ken
Chuian-Fu
CF
Institute of Marine Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, Taiwan.
Weng
De-Feng
DF
Duan
Kow-Jen
KJ
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D013482:Superoxide Dismutase