Spectral Properties of Rat Adrenal-Mitochondrial Cytochrome <em>P</em>-450.

Difference spectra produced by the binding of steroids to rat adrenal-mitochondrial cytochrome P-450 indicate distinct interactions with, respectively, pregnenolone, 11-deoxycorticosterone and 25-hydroxycholesterol. These so-called type I (deoxycorticosterone and hydroxycholesterol) or type II (pregnenolone) spectral changes have been compared in bovine adrenal-cortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were subjected to one of three pretreatments; rats were either given an ether stress to increase the plasma concentration of adrenocorticotrophic hormone or were injected with the protein-synthesis inhibitor cycloheximide, or kept in a quiescent state. These experiments substantiate the theory that distinct P-450 cytochromes are involved in cholesterol side-chain cleavage (P-450SCC) and steroid 11β-hydroxylation (P-45011β). A pH-sensitive cholesterol complex of cytochrome P-450SCC was quantitated by means of the pregnenolone difference spectrum. Adrenal mitochondria from ‘stressed’ rats had about twice as much of this cholesterol complex of cytochrome P-450SCCas adrenal mitochondria from rats kept quiescent or subjected to cycloheximide treatment. The pH-dpendence of the cytochrome P-450SCC-cholesterol complex was unaffected by the pretreatment of the animals. The type-I spectral change produced by 25-hydroxycholesterol was unaffected by stress or by changes in pH but was almost removed by sonication. The binding of 25-hydroxycholesterol to adrenal mitochondrial cytochrome P-450 was competitively inhibited by pregnenolone but was unaffected by deoxycorticosterone. The 25-hydroxycholesterol difference spectrum may detect a distinct form of cytochrome P-450SCC. Sonication of adrenal mitochondria from quiescent and cycloheximide-treated rats increased both the absorbance change produced by pregnenolone and the cholesterol side-chain cleavage activity to values approaching those fund in mitochondria from stressed rats. The rate of side-chain cleavage of added 25-hydroxycholesterol was greater than that observed with endogenous cholesterol and this rate was unaffected by prior stress to the rats. It is suggested that the low cholesterol side-chain cleavage activity in adrenal mitochondria from quiescent rats and rats given cycloheximide may be due to the existence within the mictochondrial membrane of specific ‘restraints’ upon the interaction between a certain fraction or pool of cholesterol and cytochrome P-450SCC. The availability (or restraint) of this cholesterol pool may be altered by the action of adrenocorticotrophic hormone, and mediated by a labile protein factor. [ABSTRACT FROM AUTHOR]