Purification and characterisation of rat kidney glutathione reductase.
Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2',5'-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A (273)/A (463,) A (280)/A (460), A (365)/A (460), and A (379)/A (463), were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 degrees C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q(10)) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km((NADPH)) and kcat/Km ((NADPH)) values were found to be 15.3 +/- 1.4 microM and 1.68 x 10(7) M(-1) s(-1) for the concentration range of 10-200 microM NADPH and when GSSG is the variable substrate, the Km((GSSG)) and the kcat/Km((GSSG)) values of 53.1 +/- 3.4 microM and 4.85 x 10(6) M(-1) s(-1) were calculated for the concentration range of 20-1,200 microM GSSG.
Can
Betul
B
Department of Biochemistry, Hacettepe University, Sihhiye, Ankara, Turkey.
Kulaksiz Erkmen
Gulnihal
G
Dalmizrak
Ozlem
O
Ogus
I Hamdi
IH
Ozer
Nazmi
N
D000818:Animals; D004795:Enzyme Stability; D019803:Glutathione Disulfide / Q000378:metabolism; D005980:Glutathione Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D009249:NADP; D005980:Glutathione Reductase; D019803:Glutathione Disulfide