Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography.

Y Yasukochi,B S Masters

doi:10.1016/s0021-9258(17)33166-6

Abstract

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.

Highlights

Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al
The supernatant from a 100,000 x g for 60 min centrifugation of microsomes solubilized by Renex 690 and sodium deoxycholate was applied to the Sepharose 4B column
P-450, or benzphetamine was obtained in the absence of lipid and was not subtracted from the rate obtained with the complete incubation system

Summary

Introduction

Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al A recent report by Golf et al [6] described the use of an affinity chromatography column consisting of cytochrome c bound to Sepharose 4B, but the final preparation was not homogeneous and the enzyme could be eluted upon the addition of 1 M KC1 to the equilibration buffer. It has been, only with great difficulty that purification to homogeneity has been achieved with any of these preparations and the multiple steps required have resulted in low percentage yields [1, 2]. The procedure involves the use of Sepharose 4B-bound Ne-(6..aminohexyb-adenosine 2’,5’-bisphosphate prepared by the method of Brodelius et al [7]

Methods

Results

Conclusion