Purification and partial characterization of microsomal NADH-cytochrome b₅ reductase from higher plant Catharanthus roseus

Abstract

A simple three step procedure was used to purify microsomal NADH-cytochrome b₅ (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD⁺-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b₅ from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome c via cytochrome b₅ but not to menadione.