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

Abstract

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.

Ecdysterone Biosynthesis: A Microsomal Cytochrome-P-450-Linked Ecdysone 20-Monooxygenase from Tissues of the African Migratory Locust

“…Carbon monoxide dqyerence spectrum of a reduced mixture of microsomul and mitochondrial~actions. This difference spectrum closely resembles that of mitochondria1 cytochrome P-450 from vertebrate tissues [50,51]. The large peak at 428 nm is cytochrome a3, while the peak of cytochrome P-450 is shifted to 454 nm as a consequence of the underlying cytochrome a3 trough at 445 nm, as demonstrated by Estabrook et al spectral changes.…”

Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad.

“…This possibility was examined by administration of 25 mg of aminoglutethamide to each of two rats 1 hr before injection of the labeled HDL. Aminoglutethamide is known to block side-chain cleavage of cholesterol, a step required in the production of corticosteroids (20). Prior treatment with this agent doubled the cholesterol ester/apo A-I ratio subsequently found in adrenal cells after injection of the doubly labeled HDL.…”

Suppression of estrogens with Aminoglutethimide and hydrocortisone (medical adrenalectomy) as treatment of advanced breast carcinoma: A review

“…Thompson and Siiteri (20,21), while examining the biochemical requirements of aromatization in placental microsomes, characterized this effect in more detail. Salhanick and his coworkers (11,12,22) later examined the spectral alterations of cytochrome P-450 produced by aminoglutethimide. They demonstrated the high affinity, low capacity binding site for AG on P-450 as well as the low affinity, high capacity site.…”

“…In vitro studies from several laboratories allow comparison of the relative potency of AG as an inhibitor of various hydroxylation reactions (12,13,16,17,(20)(21)(22)(23). If potency in vivo is correlated with data obtained in vitro, one would predict the following order of inhibitory action of AG: (1) aromatization (i.e.…”