The Effect of Inhibitors of Protein Synthesis on Cholesterol Side‐Chain Cleavage in the Mitochondria of Luteinized Rat Ovaries

Abstract

Conversion of cholesterol to pregnenolone, termed cholesterol side‐chain cleavage, has been studied in isolated mitochondria from luteinized rat ovaries. Cholesterol in these mitochondria in the presence of cyanoketone is converted to one product pregnenolone on addition of a suitable electron donor. The percentage conversion of total mitochondrial cholesterol to pregnenolone is comparable to that measured by the percentage conversion of [4‐14C]cholesterol to [4‐14C]pregnenolone when the label has been preincubated at 29°C for 10 min with the mitochondria. In mitochondrial incubations the depletion of endogenous cholesterol follows the production of pregnenolone. The initial rate of production of pregnenolone (0.8 nmol × mg protein−1× min−1) was maintained in incubations for 5–6 min. Addition of free cholesterol to incubations allowed the production of pregnenolone to continue at the initial rate for at least 30 min. Treatment of rats with cycloheximide 20 min prior to killing, caused a 58% inhibition of luteal mitochondrial cholesterol side‐chain cleavage and a small but significant rise in the cholesterol content of isolated mitochondria. The cycloheximide treatment did not affect the total luteal mitochondrial cytochrome P‐450 but caused a 56% decrease in the inverted type I pregnenolone‐binding spectrum, suggesting cholesterol binding to cytochrome P‐450 had been reduced. Rats treated with chloramphenicol 3 and 10 h before killing exhibited no differences in total luteal mitochondrial cytochrome P‐450 or mitochondrial cholesterol metabolism when contrasted with saline‐injected animals. Puromycin was shown to bind to the rat luteal mitochondrial cytochrome P‐450 to produce a type I1 difference spectrum and to inhibit luteal mitochondrial cholesterol side‐chain cleavage in vitro. The results are discussed in terms of a protein which is rapidly turning over and which may be synthesised extra‐mitochondrially and in some way affect the binding of cholesterol to luteal mitochondrial cytochrome P‐450.

Impact of Aging on Steroid Hormone Biosynthesis and Secretion

“…As noted above, this step is considered rate-limiting because hydrophobic cholesterol cannot rapidly diffuse through the aqueous intermembrane space of the mitochondria to support acute steroid synthesis and requires the participation of a de novo synthesized labile protein [61][62][63][64][65]. The search for the putative regulatory protein identified several candidate proteins, namely SCP2, DBI, SAP, peripheral benzodiazepine receptor (PBR), steroidogenesis inducing protein (SIP), and steroidogenic acute regulatory protein (StAR) [58].…”

“…When supplied with freely diffusible cholesterol analog (i.e., hydroxyl cholesterols), adrenocortical and testicular Leydig cells from aging rats are totally competent in producing steroids (corticosterone and testosterone). The "acute" production of steroids is dependent upon a hormone-stimulated, rapidly synthesized, cyclohexamide-sensitive and highly labile protein whose function is to transfer cholesterol from outer to inner mitochondrial membranes [53,[61][62][63]. StarD1/StAR is now identified as a putative acute regulatory protein [53,58,59,76,90,417,418].…”

Purification and Control of Bovine Adrenal Cortical Cholesterol Ester Hydrolase and Evidence for the Activation of the Enzyme by a Phosphorylation

“…The rate-limiting step in steroidogenesis is thought to be the side-chain cleavage of cholesterol to produce pregnenolone [28], and the importance of a rapidly turning over protein in this reaction has been demonstrated [20,29,30]. Work in our laboratory has shown that the rate-limiting step in the cleavage of the cholesterol side chain is the supply of cholesterol to the mitochondria1 cholesterol side chain cleavage mixed-function oxidase enzyme [29,30].…”

“…Work in our laboratory has shown that the rate-limiting step in the cleavage of the cholesterol side chain is the supply of cholesterol to the mitochondria1 cholesterol side chain cleavage mixed-function oxidase enzyme [29,30]. If mitochondrial cholesterol is being consumed for pregnenolone production it is quite possible that in the cell the major adrenal reservoir of cholesterol esters, the lipid droplets, in conjunction with cholesterol ester hydrolase play an important role in supplying the substrate, cholesterol, to the mitochondria for pregnenolone production induced by adrenocorticotropin.…”

Action of gonadotrophic hormones on cholesterol side-chain cleavage and cholesterol ester hydrolase in the ovary of the immature rat

“…The activity of the cholesterol side-chain cleavage enzyme is of particular interest because the rate of pregnenolone synthesis by this enzyme appears to limit the rate of steroidogenesis in ovarian follicles and corpora lutea (Dorrington, 1977). The major pool of cytochrome P-450 in the corpus luteum is mitochondrial (Stevenson & Taylor, 1971; Arthur & Boyd, 1974) and shows characteristics of the cytochrome P-450 that is involved in cholesterol side-chain cleavage (Jefcoate, Simpson & Boyd, 1974). The cytochrome P-450 component of the cholesterol side-chain cleavage enzyme was measured because it takes part in the binding of cholesterol, a step thought to be regulated by LH (Dorrington, 1977).…”