Black S M, Szklarz G D, Harikrishna J A, Lin D, Wolf C R, Miller W L
Department of Pediatrics, University of California, San Francisco 94143-0978.
Endocrinology. 1993 Feb;132(2):539-45. doi: 10.1210/endo.132.2.8425475.
The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein [adrenodoxin reductase (AdRed)] and an iron sulfur protein [adrenodoxin (Adx)]. The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins. Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins. These, plus purified human Adx similarly prepared by Dr. L. Vickery, were injected into rabbits to raise antiserum to each of the proteins. Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting. Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mM 8-bromo-cAMP (8Br-cAMP) or 30 nM phorbol 12-myristate 13-acetate (PMA; phorbol ester) plus 1 microM A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively. In JEG-3 cells, 8Br-cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed. The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/A23187. In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx. The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187. These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steroidogenesis in these cell systems is regulated principally at the level of mRNA abundance.
胆固醇转化为孕烯醇酮是类固醇激素合成中的限速步骤，该反应发生在线粒体细胞色素P450scc上，它通过一种黄素蛋白[肾上腺皮质铁氧化还原蛋白还原酶（AdRed）]和一种铁硫蛋白[肾上腺皮质铁氧化还原蛋白（Adx）]从NADPH接收电子来催化此反应。编码这些蛋白质的基因和mRNA的行为已在多个系统中进行了研究，但对人类蛋白质的行为了解甚少。利用克隆的人类P450scc和AdRed的cDNA，我们构建了细菌表达载体以制备毫克量的相应蛋白质。这些蛋白质，再加上由L. Vickery博士以类似方式制备的纯化的人类Adx，被注射到兔子体内以产生针对每种蛋白质的抗血清。每种抗血清都具有高度特异性，并且不会与通过蛋白质印迹法可检测到的其他线粒体蛋白质发生交叉反应。然后，将人类JEG - 3绒毛膜癌细胞和小鼠Y - 1肾上腺皮质癌细胞分别用1 mM 8 - 溴 - cAMP（8Br - cAMP）或30 nM佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA；佛波醇酯）加1 μM A23187（钙离子载体）孵育0 - 24小时，以分别激活蛋白激酶 - A和 - C途径。在JEG - 3细胞中，8Br - cAMP增加而PMA/A23187略微降低P450scc和Adx的丰度，但两种处理对AdRed均无明显影响。这些细胞产生的孕烯醇酮在8Br - cAMP作用下增加了3倍，而在PMA/A23187作用下降至三分之一。在Y - 1细胞中，8Br - cAMP增加了所有三种蛋白质的丰度，而PMA/A23187降低了P450scc和Adx的丰度。这些细胞产生的孕烯醇酮在8Br - cAMP作用下增加了9倍，并且不受TPA/A23187的影响。这些研究表明，胆固醇侧链裂解系统的三种蛋白质对8Br - cAMP和PMA/A23187的反应与对它们的基因和mRNA的研究所预测的一致，表明这些细胞系统中类固醇生成的长期调节主要在mRNA丰度水平上进行。
