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

1 Conversion of cholesterol to pregnenolone, termed cholesterol side‐chain cleavage, has been studied in isolated mitochondria from luteinized rat ovaries. 2 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‐ 14 C]cholesterol to [4‐ 14 C]pregnenolone when the label has been preincubated at 29°C for 10 min with the mitochondria. 3 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. 4 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. 5 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. 6 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.7 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.