Purification and characterization of glutathione reductase from bovine erythrocytes.
Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)(-1). For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55 degrees C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Km and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.
Erat
Mustafa
M
Biotechnology Application and Research Center, Arts and Science Faculty, Erzurum, Turkey.
Sakiroglu
Halis
H
Ciftci
Mehmet
M
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP; D013696:Temperature
D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione