Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase.

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.

• Publication year

  1986

• Venue

  Journal of Biological Chemistry

• Publication date

  1986-06-15

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(19)57476-2PMID 3086319
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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