Abstract
The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane.
Highlights
The relative concentration of cytochrome P-450 remaining in the inner membrane is shown in Fig 1 and is a clear indication that P-450 is located in the inner membrane
Our present results strongly suggest that both P-450, and P-450, are localized on the matrix side of the inner mitochondrial membrane
The variations in accessibility of P-450 to tryptic digestion in intact, sonicated, and cholate-treated inner membranes suggest that both P-450s are deeply buried in the inner membrane with a portion extending outward into the mitochondrial matrix
Summary
Adrenocortical mitochondria contain a monooxygenase system which catalyzes the conversion of cholesterol’ to pregnenolone, as well as the lip-hydroxylation of deoxycorticosterone, and the 1%hydroxylation of corticosterone (l-4) This multienzyme system is composed of an NADPH-specific flavoprotein (adrenodoxin reductase) [5, 6], an iron-sulfur protein (adrenodoxin) [7], and a specific cytochrome P-450 for the side chain cleavage and ll/?-hydroxylation reactions [8,9,10,11,12]. It is well known that the rate-limiting step of adrenal corticoidogenesis is the conversion of cholesterol to pregnenolone by the mitochondrial P-450,,,-dependent hydroxylase [21,22] This side chain cleavage reaction is dramatically
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