Properties of an adrenal cytochrome P-450 (P-450scc) for the side chain cleavage of cholesterol

doi:10.1016/0003-9861(78)90279-5

Archives of Biochemistry and Biophysics

Volume 190, Issue 1, September 1978, Pages 300-306

https://doi.org/10.1016/0003-9861(78)90279-5 Get rights and content

Abstract

A highly purified (12 nmol of P-450-heme per milligram of protein) bovine adrenal cortex mitochondrial cytochrome P-450, termed P-450_sce, which cleaves the side chain of cholesterol to yield pregnenolone, is obtained in the substrate-bound ferric form with observed absorption maxima at 393 nm and 645 nm and a shoulder around 540 nm. The absorption spectra of the P-450_scc, whether in the substrate-bound ferric form or in the CO-complexed ferrous form, are subject to environmental perturbation. The addition of adrenal ferredoxin readily restores full ferric high spin type spectrum of the substrate-bound P-450_scc or, together with cholesterol and Tween 20, restores the CO-spectrum of the P-450_scc, exhibiting stable and typical spectra of cytochrome P-450. Tween 20, at concentration of 0.3%, remarkably increases the P-450_scc-catalyzed cholesterol side chain cleavage activity. Based on these findings, a highly reactive and reliable assay has been developed for the conversion of cholesterol to pregnenolone. The specific activity of the P-450_scc, thus determined in the presence of NADPH, NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1), adrenal ferredoxin, cholesterol, and molecular oxygen, is 16 mol of pregnenolone formed per minute per mole of P-450-heme and V of enzyme catalyzed reaction was 30 mol/min/mol of P-450-heme. Apparent K_m values are 120 μm for cholesterol and 1.5 μm for adrenal ferredoxin. The P-450_scc has a pH optimum at pH 7.2 and is most active at ionic strength of 0.1.

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Since PLs seem to be essential for a stimulatory effect of drugs on CYP11A1, we carried out spectral titrations of this P450 under the conditions modeling those in the enzyme assay: 10:1 (M/M) ratio of cholesterol to CYP11A1 but a 4-fold increased substrate and enzyme concentrations to obtain good quality spectra. Consistent with previous studies (Takikawa et al., 1978), cholesterol-induced spectral shifts in CYP11A1 were of type I (Fig. 3A and C) occurring when compound in question binds to the P450 active site and displaces water molecule coordinating the heme iron in substrate-free enzyme. Subsequent titration of cholesterol-bound CYP11A1 with either pemirolast, clobenpropit, desogestrel, dexmedetomidine or tizanidine revealed that, as in experiments with substrate-free CYP11A1, only clobenpropit and dexmedetomidine elicited spectral changes in substrate-bound CYP11A (Figs. 3A and D and 4A and D).
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The generally accepted version (GAV) of the chemical processes by which the steroid hormones are biosynthesized cannot be considered to be an inerrant description of in vivo processes. Customarily this version is derived by piecing together the results obtained from several independent artificial in vitro incubation experiments. Extrapolation of such results from in vitro to in vivo requires untested assumptions which introduce varying degrees of uncertainty. In vitro incubation experiments reveal only what is possible; not what actually prevails in situ. Presented here are hypothetical alternative renditions of some of the oxidative processes involved in steroidogenesis. These versions suggest that some cytochrome P-450's catalyze the introduction of both oxygen atoms of dioxygen into an appropriate sterol precursor. The products are conceived as oxygen free radicals (peroxy or 1,2-cyclic peroxy) which serve as the “reactive intermediates” (the precursors) for the hormones. The true intermediates are not stable, isolable, hydroxylated compounds as they are customarily portrayed in the GAV. Central to these new renditions is the hypothesis that the appropriate P-450 introduces dioxygen into the precursor yielding either: A, a 20 peroxy sterol species or B, a species oxygenated at both C-17 and C-20 or C, a species oxygenated at both C-20 and C-21. In this hypothesis, A would serve as the precursor for progesterone, B, for the C₁₉-androgens and C₁₈-estrogens and C, for the mineralocorticoids (corticosterone and aldosterone) and the glucocorticoid (cortisol). How this version of steroidogenesis can be used to understand the etiologies of various genetically derived enzyme deficiency diseases of the adrenal and ovaries will be discussed. If as proposed here, the various polyfunctional cytochromes (P-450_scc, P-450_c17, P-45011B1 (P-450_cortisol), P-45011B2 (P-450_aldo), etc.) catalyze conversions that are different from simple hydroxylations, the labels usually given these deficiency diseases may not be appropriate. More importantly, these new conceptions may clarify the etiology of some of the characteristic symptoms of these diseases that are not now adequately explained by the GAV.
Early years of oxygenase research in Bethesda, Osaka, Urbana, and Kanazawa
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Under a newly developed sensitive assay system, the purified P450scc preparation specifically catalyzed the side-chain cleavage of cholesterol to form pregnenolone with a turnover number of 16 min−1 in the presence of adrenodoxin and NADPH-adrenodoxin reductase, but did not catalyze 11β- or 18-hydroxylase activity for deoxycorticosterone. In contrast, P45011β catalyzed 11β- and 18-hydroxylase activities for deoxycorticosterone with turnover numbers of 110 and 18 min−1, respectively [27,28]. Each rabbit antibody produced against P450 interacted with respective P450, but not with alternative adrenal cortex mitochondrial P450 or P450cam.
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Citation Excerpt :
Anaerobic measurements were carried out under an argon atmosphere. The reaction buffer for CYP11A1 consisted of 50 mm potassium phosphate (pH 7.3), 0.1 m KCl, 0.1 mm EDTA, 0.1 mm dithioerythritol, and 0.05% sodium cholate (35). Syringe A contained 2 μmP450scc and 40 μm cholesterol.
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^☆: This is paper II in a series. The accompanying papers are Ref. 1 (I) and Ref. 2 (III). This investigation was supported by Grants 143030, 178055, and 268051 from the Ministry of Education, Science and Culture, Japan, and the Naito Research Grant for 1975.

²: Present address: Institute for Protein Research, Osaka University, Suita 565, Japan.

³: Present address: Department of Environmental Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan.

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Properties of an adrenal cytochrome P-450 (P-450scc) for the side chain cleavage of cholesterol☆

Abstract

Arch. Biochem. Biophys

Arch. Biochem. Biophys

J. Biol. Chem

Biochim. Biophys. Acta

J. Biol. Chem

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Arch. Biochem. Biophys

Properties of an adrenal cytochrome P-450 (P-450_scc) for the side chain cleavage of cholesterol☆