The effect of glycerol in the storage medium on the inhibitory kinetic behavior of glutathione reductase

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°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 μM) and Ki(NADP+) (26.6 ± 2.2 μM) 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 μM) and linear mixed-type (Ki = 59.6 ± 8.4 μM; Ks = 49.9 ± 3.2 μM; α = 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)