Tuckey R C, Holland J W
Raine Center for the Study of Perinatal and Developmental Biology, Department of Biochemistry, University of Western Australia, Nedlands, Perth.
J Biol Chem. 1989 Apr 5;264(10):5704-9.
Substrate turnover rates by cytochrome P-450scc were measured in mitochondria isolated from corpora lutea and granulosa cells of follicles. Hydroxycholesterol substrates were added to the mitochondria to test the degree of saturation of the cytochrome with endogenous cholesterol during pregnenolone synthesis. 25-Hydroxycholesterol proved unsuitable for this since it was converted into pregnenolone with a maximum velocity of only 25% of that for cholesterol. 20 alpha-Hydroxycholesterol was found to be suitable providing correction was made for the one less hydroxylation required to convert this substrate into pregnenolone, compared to cholesterol. Mitochondria isolated from large follicles and corpora lutea displayed biphasic time courses for pregnenolone synthesis from endogenous cholesterol with a rapid phase lasting for 2-4 min and a slow phase which was linear for at least 30 min. Only a single rapid phase was observed for these mitochondria in the presence of 20 alpha-hydroxycholesterol. From the degree of stimulation of the substrate turnover rate by this steroid, it was concluded that the endogenous cholesterol concentration was saturating during the fast phase for large follicles but subsaturating in luteal mitochondria. Time courses for pregnenolone synthesis by mitochondria isolated from granulosa cells of small and medium follicles were linear for 30 min and gave a substrate turnover rate of 16-18 mol of steroid/min/mol of cytochrome P-450scc, similar to the turnover rates under saturating substrate conditions determined for large follicles and corpora lutea. The substrate turnover rate for cytochrome P-450scc in medium follicles was not increased by the addition of 20 alpha-hydroxycholesterol, indicating that the cholesterol concentration in the steroidogenic pool of these mitochondria was saturating and remained so over the 30-min duration of the incubation. It is therefore unlikely that gonadotropin stimulation of granulosa cells of small to medium follicles could acutely regulate pregnenolone synthesis by increasing the rate of transfer of cholesterol into a steroidogenic pool. This study shows that as the cytochrome P-450scc concentration in porcine ovarian mitochondria increases during follicular growth and luteinization there is a decrease in the fractional saturation of the cytochrome with cholesterol.
测定了从黄体和卵泡颗粒细胞分离的线粒体中细胞色素P-450scc的底物周转率。将羟胆固醇底物添加到线粒体中，以测试孕烯醇酮合成过程中细胞色素与内源性胆固醇的饱和程度。结果证明25-羟胆固醇不适合用于此实验，因为它转化为孕烯醇酮的最大速度仅为胆固醇的25%。发现20α-羟胆固醇是合适的，但与胆固醇相比，将该底物转化为孕烯醇酮所需的羟基化少一个，因此需要进行校正。从大卵泡和黄体分离的线粒体，以内源性胆固醇合成孕烯醇酮呈现双相时间进程，快速相持续2-4分钟，慢速相至少30分钟呈线性。在存在20α-羟胆固醇的情况下，这些线粒体仅观察到一个快速相。根据该类固醇对底物周转率的刺激程度得出结论，大卵泡快速相期间内源性胆固醇浓度处于饱和状态，但黄体线粒体中不饱和。从小卵泡和中卵泡颗粒细胞分离的线粒体合成孕烯醇酮的时间进程在30分钟内呈线性，底物周转率为16-18摩尔类固醇/分钟/摩尔细胞色素P-450scc，类似于在大卵泡和黄体饱和底物条件下测定的周转率。添加20α-羟胆固醇不会增加中卵泡中细胞色素P-450scc的底物周转率，表明这些线粒体类固醇生成池中胆固醇浓度处于饱和状态，并且在孵育的30分钟内一直保持饱和。因此，促性腺激素刺激小到中卵泡的颗粒细胞不太可能通过增加胆固醇向类固醇生成池的转移速率来急性调节孕烯醇酮的合成。这项研究表明，随着猪卵巢线粒体中细胞色素P-450scc浓度在卵泡生长和黄体化过程中增加，细胞色素与胆固醇的分数饱和度会降低。
