Spectral Properties of Rat Adrenal‐Mitochondrial Cytochrome P ‐450

Difference spectra produced by the binding of steroids to rat adrenal‐mitochondrial cytochrome P ‐450 indicate distinct interactions with, respectively, pregnenolone, 11‐deoxycorti costerone and 25‐hydroxycholesterol. These so‐called type I (deoxycorticosterone and hydroxy‐cholesterol) or type II (pregnenolone) spectral changes have been compared in bovine adrenalcortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were 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 ‐450 SCC ) and steroid 11β‐hydroxylation ( P ‐450 11β ). A pH‐sensitive cholesterol complex of cytochrome P ‐450 SCC 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 ‐450 SCC as adrenal mitochondria from rats kept quiescent or subjected to cycloheximide treatment. The pH‐dependence of the cytochrome P ‐450 SCC ‐cholesterol complexs 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 ‐450 SCC . 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 found 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 mitochondrial membrane of specific “restraints” upon the interaction between a certain fraction or pool of cholesterol and cytochrome P ‐450 SCC . The availability (or restraint) of this choleterol pool may be altered by the action of adrenocorticotrophic hormone, and mediated by a labile protein factor.