Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property.
A full-length cDNA of 803 base pairs encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Epinephelus malabaricus was cloned by the polymerase chain reaction 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 (65-91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-49, -64, and -121) and zinc (His-64, -72, and -81 and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, was well-conserved among all reported Cu/Zn-SOD sequences. To further characterize the E. malabaricus Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+) and transformed into Escherichia coli BL21(DE3)pLysS. 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. The enzyme activity was inhibited under basic pH (higher than 10.0). The enzyme retained 65% activity after heating at 60 degrees C for 10 min. The inactivation rate constant (k(d)) was 6.64 x 10(-2) min(-1) at 60 degrees C. The enzyme activity was only some decrease under 3% sodium dodecyl sulfate. The enzyme was resistant to proteolysis by trypsin and chymotrypsin. The finding of Cu/Zn-SOD cDNA could be used as a probe to detect the transcription level of this enzyme, which can be used as an early biomarker of environmental pollution. The property of this enzyme could provide a reference as compared to the oxidized forms or new isoforms, which could be induced under the experiments of pollution.
Ken
Chuian-Fu
CF
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 202, ROC.
Cheng
Yu-Feng
YF
Chang
Ching-Fong
CF
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010473:Perciformes / Q000235:genetics*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D013482:Superoxide Dismutase