Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro.
Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2',5'-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K(m) values for G6-P and NADP(+) were determined to be 0.026, 0.0068 mM, respectively. The V(max) values for G6-P and NADP(+) were 2.20 and 2.27 μM min(-1) mg protein(-1), respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10(-6) min(-1) mg protein(-1), respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC50 values of Zn(+2), Mn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn(+2), Al(+3), and Cd(+2) showed competitive inhibition, while Cu(+2) inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.
Fishery College, Huazhong Agricultural University, Wuhan 430070, China.
Wei
W
Hu
Luo
L
Zhi
Mei-Qin
MQ
Zhuo
Qing-Ling
QL
Zhu
Jia-Lang
JL
Zheng
Qi-Liang
QL
Chen
Yuan
Y
Gong
Cai-Xia
CX
Liu
D000818:Animals; D002347:Carps / Q000378:metabolism*; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D043143:Hepatopancreas / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D019216:Metals, Heavy / Q000633:toxicity*; D013696:Temperature
D019216:Metals, Heavy; D005954:Glucosephosphate Dehydrogenase