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

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 chromatograph...

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Bibliographic Details
Published in:	Biochemical and biophysical research communications Vol. 197; no. 2; p. 518
Main Authors:	Madyastha, K M, Chary, N K, Holla, R, Karegowdar, T B
Format:	Journal Article
Language:	English
Published:	United States 15-12-1993
Subjects:	Chromatography, Affinity Cytochrome Reductases - isolation & purification Cytochrome Reductases - metabolism Cytochrome-B Reductase Electrophoresis, Polyacrylamide Gel Kinetics Microsomes - enzymology Molecular Weight Plants - enzymology
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Summary:	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 microM), 2, 6-dichlorophenolindophenol (Km 65 microM) and cytochrome c via cytochrome b5 but not to menadione.
ISSN:	0006-291X
DOI:	10.1006/bbrc.1993.2509