@article{oai:omu.repo.nii.ac.jp:00009375,
 author = {稲葉, 俊夫},
 journal = {Bulletin of the University of Osaka Prefecture. Ser. B, Agriculture and biology},
 month = {Mar},
 note = {application/pdf, In the mammarian ovary, after ripening and rupture of follicle, there is formed a corpus luteum. The corpus luteum secrets progesterone whose main action is connected with preparation for and maintenance of pregnancy. Although luteinizing hormone (LH) and prolactin are known to stimulate the production of progesterone in the corpus luteum, these mechanism are not well understood. The rate-limiting step of steroido-genesis in steroid-hormone-producing tissues is thought to be cholesterol side-chain cleavage (CSCC, HALL & YOUNG, 1968). The CSCC enzyme complex is considered to be a most probable locus for gonadotropic regulation of steroidogenesis in corpora lutea, although other enzymes and membrane sites have not been excluded. Beyond the initial gonadotropic impact, i.e., cAMP formation, the only recognized pathway for activation of steroidogenesis is via cAMP-dependent protein kinase. In the present investigation, the regulatory role of cAMP-dependent protein kinase on steroidogenesis has been studied in luteal cell mitochondria prepared from heavily luteinized prepubertal rat ovaries. Studies on the subcellular distribution of protein kinase activity in the corpus luteum obtained from PMS-hCG treated rats revealed that 72.4±3.4% of total cellular protein kinase activity was in the cytosol fractions, and 5.5±0.1% of that was in the mitochondrial fractions, as cAMP-dependent. Preliminary to more extensive studies of intracellular compartments as loci for the regulation of steroidogenesis, tightly coupled mitochondria from rat ovaries have been prepared by differential centrifugation. Morphological and functional intactness of these luteal cell components was demonstrated by electron microscopy and by having tightly coupled oxidative phosphorylation systems in their respiration. The major steroid products isolated, following incubation of mitochondria with labeled free cholesterol and L-malate, were pregnenolone and progesterone, which were characterized by chromatography and carrier recrystallization analysis. Progesterone synthesis by luteal mitochondria indicated not only that these organelles contained the CSCC enzyme system but that they also contained 3β-hydroxysteroid dehydrogenase-isomerase complex. To permit a more direct exploration of mitochondrial steroidogenesis, methods for radioimmunoassay of pregnenolone have been studied. Antiserum was generated in a rabbit against pregnenolone-16α-carboxyethyl thioether conjugated to bovine serum albumin. The antibody, used for the assay of pregnenolone in extracts of serum and tissue homogenates, proved sufficiently specific to allow direct assay of extracts without chromatography. The accuracy and precision of the assay method for pregnenolone were satisfactory. Using the radioimmunoassay method, net pregnenolone production in luteal cell mitochondria was determined. In the presence of 17β-hydroxy-4, 4, 17α-trimethylandrost-5-eno-(2, 3-d)-isoxazole and a suitable electron donor, cholesterol was converted to a single product, i.e., pregnenolone, in luteal cell mitochondria. Mitochondria secreted more pregnenolone when incubated with cAMP or partially purified cytosol protein kinase in the presence of 1mM CaCl_2. In the absence of Ca^<++>, however, cAMP and protein kinase stimulation was not observed, suggesting the presence of a permeability barrier excluding extramitochondrial protein kinase from the CSCC enzyme system. To clarify the possibility that Ca^<++> is involved in LH induced intramitochondrial activation, the partially purified cytosol protein kinase was added to a solubilized preparation of the CSCC enzyme complex obtained from luteal cell mitochondria. After 30min incubation, a stimulation of pregnenolone production was observed, suggesting that Ca^<++> was not required for the direct stimulation of protein kinase on the solubilized CSCC enzyme complex. In the present study, it seems that Ca^<++> facilitates the access of protein kinase to the CSCC enzyme complex in the mitochondrion. It is observed that an interval of only 15 minutes occurred between the time of LH administration to rats and the demonstrable increase in progesterone production by ovary. If cholesterol is utilized by the CSCC enzyme complex which is located on the matrix side of the inner membrane, cholesterol moleclues have to migrate across the mitochondrial membranes. The rate of "flip-flop" of cholesterol molecules in phospholipid bilayers, however, is known to be slow with a half-life of more than 1 hour in most cases. In order to explain the rapid response of LH, mitochondria were preincubated without sterol substrate. The addition of cAMP and cytosol protein kinase to cholesterol-depleted mitochondria caused a significant increase in pregnenolone production in the presence of Ca^<++>, suggesting that protein kinase could stimulate cholesterol supply to the CSCC enzyme system. It is concluded from these results that protein kinase stimulates cholesterol conversion to pregnenolone and progesterone in luteal cell mitochondria in the presence of Ca^<++>, probably through the stimulation of cholesterol supply to the CSCC enzyme complex., Bulletin of the University of Osaka Prefecture. Ser. B, Agriculture and biology. 1983, 35, p.135-165},
 pages = {135--165},
 title = {ラット黄体ミトコンドリアのCholesterol側鎖断裂酵素に対するProtein Kinaseの作用機序},
 volume = {35},
 year = {1983}
}