The effect of glycerol in the storage medium on the inhibitory kinetic behavior of glutathione reductase
2012
Kulaksiz-Erkmen, G., Hacettepe University, Sihhiye-Ankara (Turkey). Faculty of Medicine | Can, B., Hacettepe University, Sihhiye-Ankara (Turkey). Faculty of Medicine | Dalmizrak, O., Hacettepe University, Sihhiye-Ankara (Turkey). Faculty of Medicine | Ogus, I.H., Hacettepe University, Sihhiye-Ankara (Turkey). Faculty of Medicine | Ozer, N., Hacettepe University, Sihhiye-Ankara (Turkey). Faculty of Medicine
In this study we report on the effect of glycerol used in storage buffers, on the inhibitory kinetic behavior of NADP+ on rat kidney glutathione reductase (rkGR) stored at -80 deg C in +/- 10% (v/v) glycerol. At fixed [GSSG] and varied [NADPH], rkGR stored +/- glycerol were inhibited competitively by NADP+. Although the addition of glycerol decreased Km (9.7 +/- 0.9 microM) and Ki(NADP+) (26.6 +/- 2.2 microM) for NADPH, the catalytic efficiency (kcat/Km = 2.75 x 107 M-1.s-1) was increased 1.64 fold. At fixed [NADPH] and varied [GSSG], rkGR exhibited noncompetitive (Ki = 397 +/- 69 microM) and linear mixed-type (Ki = 59.6 +/- 8.4 microM; Ks = 49.9 +/- 3.2 microM; alpha = 7.74) inhibitions with and without glycerol during storage. The kcat/ Km of rkGR stored in glycerol was 5.16 x 106 M-1.s-1. Although the increase in kcat was 1.25 fold, the kcat/Km was minimally affected (1.06).
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