Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation.

N.J Greenfield,B Gerolimatos,B.S Szwergold,A.J Wolfson,V.V Prasad,S Lieberman

doi:10.1016/s0021-9258(19)69449-4

Abstract

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.

Highlights

From the Department of Biochemistry, the Department of Obstetrics and Gynecology and the International Znstitute for the Study of Human Reproduction, The College of Physicians and Surgeons, CoZumbia Uniuersity, New York, New York 10032
Chromatography of the octyl Sepharose eluate on cholesterol acetate (CA)-Sepharose removed extraneous proteins and resolved the cytochrome P-450, Evidence has recently been presented that supports the idea that mitochondria from bovine adrenal cortex contain more than one cholesterol’ side chain cleavage enzyme system [1].The adrenalglands contain three types of steroidal intermediates: free, i.e. unesterified steroids(such as pregnenointo two fractions, each of which displayed monophasic lone), the steroidal 3-sulfates such as pregnenolone sulfate binding with all three substrates
The results of Wolfson and Lieberman [1] indicated that there may bepresent in sonicated mitochondria frombovine adrenal cortex more than oneenzyme system that catalyzes the oxidative cleavage of the side chain of cholesterol

Summary

Introduction

Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450,, Evidence has recently been presented that supports the idea that mitochondria from bovine adrenal cortex contain more than one cholesterol’ side chain cleavage enzyme system [1].The adrenalglands contain three types of steroidal intermediates: free, i.e. unesterified steroids(such as pregnenointo two fractions, each of which displayed monophasic lone), the steroidal 3-sulfates such as pregnenolone sulfate binding with all three substrates These fractions be- and dehydroisoandrosterone sulfate, and the recently identihaved identically with respect to their ability to bind fied lipoidal derivatives of steroids [2, 3]. Kinetic experiments using sonicated mitochondria asthe enzyme source revealed that each substrate exhibited two K , values

Methods

Results

Conclusion