| Loerwald, B; Clemens, S; Barz, W: Purification and characterization of the NADPH-cytochrome P-450 (cytochrome C) reductase from chickpea, Journal of Plant Physiology, 149 (Issue 5), 633-635 (1996), doi:10.1016/S0176-1617(96)80347-6 | |
| Abstract: The membrane-bound flavoprotein NADPH-cytochrome P-450 (cytochrome c) reductase, which functions in electron transfer to cytochrome P-450 monooxygenases, was solubilized by detergent from microsomal fractions of chickpea cell cultures and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2′, 5′-bisphosphoadenosine-Sepharose 4B. An apparent molecular mass of 85 kDa was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme followed Michaelis-Menten kinetics with Km, values of 11.4μmol/L for NADPH and 7.5μmol/L for cytochrome c. The reductase was competitively inhibited by NADP+ (Ki = 16μmol/L). Isoelectric focusing of the purified reductase led to an pI of 5.9. The purified reductase showed higher activity after incubation with equimolar quantities of FAD and FMN. |
