Bakker G H, Hoogerbrugge J W, Rommerts F F, van der Molen H J
Biochem J. 1981 Aug 15;198(2):339-46. doi: 10.1042/bj1980339.
Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.
肿瘤性睾丸间质细胞在有或无促黄体素（促黄体生成素，LH）的情况下进行孵育。在1 - 甲基 - 3 - 异丁基黄嘌呤（0.25mmol/L）存在时，用促黄体素（1000ng/ml）刺激细胞，会导致类固醇生成增加以及蛋白质磷酸化增加。当孕烯醇酮代谢被抑制时，基础孕烯醇酮生成量为每10⁶个细胞26.9±7.4ng/60分钟；刺激后的生成量为每10⁶个细胞156.1±39.5ng/60分钟（平均值±标准差，n = 4）。检测到分子量为17000、22000、24000、33000和57000Da的促黄体素依赖性磷酸化蛋白。观察到伴随着孕烯醇酮生成增加，这些磷酸化蛋白中[(³²)P]Pi掺入量显著增加。研究了这些磷蛋白在细胞核、线粒体和线粒体后上清液中的存在情况。17000Da的磷蛋白存在于细胞核部分，而22000、24000、33000和57000Da的磷蛋白定位于线粒体后上清液部分。从肿瘤性睾丸间质细胞分离的线粒体部分中存在80.3±6.1%（平均值±标准差，n = 5）的胆固醇侧链裂解活性，当细胞用促黄体素/1 - 甲基 - 3 - 异丁基黄嘌呤预孵育后，该活性增加了2.5倍（基础生成量：每毫克蛋白质194.6±28.6ng/30分钟；促黄体素刺激后的生成量：每毫克蛋白质498.8±91.5ng/30分钟；平均值±标准差，n = 3）。添加促黄体素/1 - 甲基 - 3 - 异丁基黄嘌呤后蛋白质磷酸化动力学与孕烯醇酮生成动力学的相似性表明，磷蛋白可能参与肿瘤性睾丸间质细胞中促黄体素依赖性的类固醇生成增加。然而，线粒体外部的磷蛋白在多大程度上能影响线粒体内的胆固醇侧链裂解活性仍有待证明。
