Substrate-induced difference spectra and cholesterol to pregnenolone conversion with adrenal heme protein P-450

Burstein, Shlomo; Co, Nana; Gut, Marcel; Schleyer, Heinz; Cooper, David Y.; Rosenthal, Otto

doi:10.1021/bi00754a015

Cited by 15 publications

(3 citation statements)

References 13 publications

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“…In our preparations type I spectra were not produced by the addition of cholesterol to the membrane-bound P-450, although several other laboratories have demonstrated such spectra with cholesterol-depleted cytochrome P-450 from the adrenal cortex (Burstein et al, 1972;Harding et al, 1971). This implies that the substrate binding site of mitochondrial P-450 is saturated with endogenous cholesterol and is consistent with the theory that the RT I spectrum results from the displacement of bound substrate cholesterol (Schenkman et al, 1972).…”

Section: Discussionmentioning

confidence: 52%

Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation: an assay of enzyme-specific inhibitors

Uzgiris¹,

Graves²,

Salhanick³

1977

Biochemistry

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Absorbance changes in the spectrum of cytochrome P-450 were related to the inhibition of [26-14C]cholesterol oxidation to [14C]isocaproate and pregnenolone in mitochondria from bovine corpus luteum produced by two types of ligands. Nitrogenous inhibitors, such as aminoglutethimide, elicit an absorption maximum at about 427 nm and a minimum at about 393 nm (type II), while steroidal inhibitors, such as (20R)-20-(p-tolyl)-5-pregnene-3beta,20-diol (20-tolyl-pregnenediol), cause difference spectra with maximum at about 420 nm and minimum at about 390 nm (reverse type I). The magnitude of spectral change and the amount of inhibition of pregnenolone synthesis by aminoglutethimide are closely correlated at concentrations ranging from 5 to 750 muM and by the model steroid, 20-tolyl-pregnenediol, at concentrations from 0.5 to 25 muM. The responses are concentration dependent and linear over the range of effective concentrations. The concentrations of inhibitors for the half-maximal inhibition of pregnenolone biosynthesis are identical with the concentrations producing half-maximal spectral changes within experimental error. Displacement of substrate from cytochrome P-450 and/or stabilization of the redox potential subsequent to to the ligation of heme iron is proposed as the specific mechanism of cholesterol side chain cleavage inhibition. Finding, together, the two procedures offer a sensitive, specific, and accurate means of screening inhibitors of the cholesterol side chain cleavage system.

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Section: Discussionmentioning

confidence: 52%

Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation: an assay of enzyme-specific inhibitors

Uzgiris¹,

Graves²,

Salhanick³

1977

Biochemistry

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“…Three fundamentally empi rical types of spectra have been well described and characterized with various drugs and other chemicals but are poorly understood: 'type F, with spectral maxima about 385 or 395 nm and minima about 420 nm; 'type IT, with minima about 390 or 410 nm and maxima about 427-430 nm; and 'reverse type 1', which is almost exactly the mirror image of type 1 spectra (21,58,67). This technique reveals nothing, how ever, in absolute terms about the changes oc curring at or near the enzyme active-sites of 2 The abbreviation 'P-450' refers to the six or more distinct forms of liver microsomal cytochrome P-450 having slightly different spectral, catalytic, electro phoretic, immunochemical and/or paramagnetic prop erties (1,17,18,47,70). Other abbreviations used include: 6 S, liver microsomal cytochrome b f \ phéno barbital microsomes, liver microsomes derived from an animal previously treated with phénobarbital in vivo; (3-naphthoflavone microsomes, liver microsomes from an animal previously treated with (J-naphthoflavone in vivo; 3-methylcholanthrcne microsomes, liver micro somes from an animal previously treated with 3-methylcholantlirene in vivo; and EPR, electron para magnetic resonance.…”

mentioning

confidence: 99%

“…When the same concentration of the same test com pound is added to microsomes, there are vari ations in whether a type I, type II, or reverse type I spectrum (or no detectable spectrum) occurs, in the shapes of the difference spectral curves, and in the peak-to-trough heights per nanomol of P 450. These apparent dis crepancies appear to reflect differences in: species (64); strains (14); age (6); tissues (9,34,49); prior treatment in vivo (25,26,28,62), including, for example, Oxytetracycline in the diet (25); techniques used during preparation of the microsomes (2,17); and prior additions in vitro (12, 13, 23, 28, 29, 49, 6 2 -6 4 , 67, 75). Even stressed rats are different from quiescent rats when steroid binding to adrenal mito chondrial P-450 was studied (29).…”

mentioning

confidence: 99%

Spectral Evidence for Weak Ligand in Sixth Position of Hepatic Microsomal Cytochrome P-450 Low Spin Ferric Iron <i>in viv</i><i>o</i>

Kumaki

Nebert

1978

Pharmacology

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The spin state of cytochrome P450 ferric iron in vivo and in vitro was examined in freshly prepared liver microsomes from rabbit, guinea pig, hamster, mouse, and rat by room-temperature difference spectrophotometry. The administration of compounds which induce certain forms of P450 (phenobarbital, β-naphthoflavone, or 3-mefhylcholanthrene) to each of these species produces consistent detectable changes in the spin state of P-450 iron in vivo. Simple liquids of known relatively ‘pure’ type I, reverse type I, or type II character (cyclohexane, butanol-1, and octylamine-1, respectively) produce detectable changes in the in vitro spin state of P450 iron. By the method of off-balance spectrophotometry (in which microsomal cytochrome b₅ is made equal in both cuvettes), we show that these differences in spin state in vivo and in vitro vary among the five mammalian species and are altered by prior treatment in vivo with the inducers of P450 and by addition in vitro of such chemicals as cyclohexane, butanol-1, and octylamine-1. The magnitude of the peak-to-trough height in type I, reverse type I, and type II difference spectra (or whether any spectrum is detected) is shown to depend largely on the in vivo spin state of the P450 iron in freshly prepared microsomes at the start of the experiment. The presence of a 410-nm trough in the type II difference spectrum is shown to represent a ligand having reverse type I character in the sixth coordination site of in vivo low spin iron. Our data therefore indicate that the sixth ligand for in vivo low spin P-450 ferric iron is a hydroxyl group from an adjacent amino acid residue of the P-450 protein (or moiety of similar ligand field strength, such as a water molecule) rather than a stronger ligand such as one having lone pair electrons from an N atom.

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Inhibition of cholesterol side-chain cleavage by 22-azacholesterol

Masry

Fee

Masry

et al. 1977

Biochemical Pharmacology

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The side-chain of cholesterol is oxidatively cleaved by a cytochrome P-450-dependent enzyme system present in mammalian adrenal mitochondria. The reaction was postulated to proceed via one or more hydroxylated intermediates. Inhibition of the reaction by the potent inhibitor 22-azacholesterol did not result in a build-up of the postulated intermediates: (ZOS)-20-hydroxycholesterol. (22R)-22.hydroxycholesterol, (20R,22R)-20,22-dihydroxycholesterol or (20S)-cholesterol hydroperoxide. Spectral studies indicate a modified type II spectrum for the reaction of 22-azacholesterol with adrenal mitochondrial cytochrome P-450. In addition, spectral and electron spin resonance studies indicate a common interaction of cholesterol and 22-azacholesterol with the cytochrome. Although this study does not resolve the question of the intermediacy of the oxygenated derivatives in the cleavage reaction. it indicates that the binding of 22-azacholesterol depends on its steroid structure rather than on its amine character. A discussion of the proximity of the heme to the substrate binding site is also presented.

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Substrate-induced difference spectra and cholesterol to pregnenolone conversion with adrenal heme protein P-450

Cited by 15 publications

References 13 publications

Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation: an assay of enzyme-specific inhibitors

Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation: an assay of enzyme-specific inhibitors

Spectral Evidence for Weak Ligand in Sixth Position of Hepatic Microsomal Cytochrome P-450 Low Spin Ferric Iron <i>in viv</i><i>o</i>

Inhibition of cholesterol side-chain cleavage by 22-azacholesterol

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