Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity.
Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout (Oncorhynchus mykiss) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, KM and Vmax values for GSSG and NADPH were also determined for the enzyme. In addition, Ki values and inhibition types were determined for GSH and NADP+. Additionally, inhibitory effects of metal ions (Cd+2, Cu+2, Pb+2, Hg+2, Fe+3 and Al+3) on glutathione reductase were investigated. Ki constants and IC50 values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. IC50 values of Cd+2,Cu+2, Pb+2, Hg+2, Fe+3 and Al+3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the Ki constants for Cd+2 and Cu+2 were 0.104+/-0.001, 0.117+/-0.001, respectively.
Tekman
Bilge
B
Atatürk University, Arts and Science Faculty Department of Chemistry, TR-25240, Erzurum-Turkey.
Ozdemir
Hasan
H
Senturk
Murat
M
Ciftci
Mehmet
M
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D009994:Osmolar Concentration; D011489:Protein Denaturation; D013696:Temperature; D014874:Water Pollutants, Chemical / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D008670:Metals; D014874:Water Pollutants, Chemical; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione; D019803:Glutathione Disulfide