Purification and Characterisation of Rat Kidney Glutathione Reductase


Can B., KULAKSIZ ERKMEN G. , DALMIZRAK Ö. , ÖĞÜŞ İ. H. , ÖZER N.

PROTEIN JOURNAL, cilt.29, ss.250-256, 2010 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 29 Konu: 4
  • Basım Tarihi: 2010
  • Doi Numarası: 10.1007/s10930-010-9246-4
  • Dergi Adı: PROTEIN JOURNAL
  • Sayfa Sayıları: ss.250-256

Özet

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 A 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 +/- A 1.4 mu M and 1.68 x 10(7) M-1 s(-1) for the concentration range of 10-200 mu M NADPH and when GSSG is the variable substrate, the Km((GSSG)) and the kcat/Km((GSSG)) values of 53.1 +/- A 3.4 mu M and 4.85 x 10(6) M-1 s(-1) were calculated for the concentration range of 20-1,200 mu M GSSG.