ANALYSIS OF THE MECHANISM OF CHOLESTEROL SIDE CHAIN CLEAVAGE REACTION AND ITS RELATED HEREDITARY DISEASES

类固醇激素在高等动物的正常生活活动中起着重要的作用，这些激素的失调会导致动物的严重疾病。类固醇激素生物合成的限速步骤是线粒体细胞色素P-450scc催化的胆固醇侧链断裂反应。绒毛膜类脂性肾上腺增生，又称Prader综合征，被认为是细胞色素P-450scc缺乏所致。然而，本研究的代谢者安田报道，患者细胞色素P-450scc的cDNA与正常人没有区别，细胞色素P-450scc蛋白实际上在患者的睾丸中表达。Miller和他在美国的同事也报道了患者肾上腺素还原酶(AR)和肾上腺素(AD)的cDNA与正常人以及细胞色素P-450scc的cDNA没有什么不同。从这些研究来看，胆固醇侧链断裂反应减弱的原因是…更多的HED在Prader综合征患者中的作用尚不清楚。在本研究中，我们试图通过酶学技术来阐明AR、AD和细胞色素P-450scc之间的相互作用导致活性降低的机制。对AR、AD和细胞色素P-450scc的印迹分析表明，患者睾丸中这些酶组分的含量与正常人没有太大差异。患者睾丸线粒体胆固醇侧链断裂活性为正常人的35%，提示患者睾丸线粒体的活性并非总是零水平。患者睾丸线粒体磷脂/蛋白比值为0.380，而正常人为0.659，表明患者睾丸线粒体磷脂明显减少。从患者睾丸线粒体中提取磷脂，分析磷脂亚类的含量。我们发现，患者睾丸线粒体中溶血磷脂的含量是正常人的7倍。已知溶血磷脂可以抑制线粒体中胆固醇侧链的断裂活性。我们还用从牛肾上腺皮质纯化的AR、AD和细胞色素p-450scc证实了溶血磷脂在重组系统中的抑制作用。这些结果表明，患者睾丸线粒体中溶血磷脂水平的升高与先天性类脂肾上腺增生症活性降低的机制有关。较少

Steroid hormones play an important role in normal life activity for higher animals, and the disorders of these hormones result in the severe diseases for the animal. The ratelimiting step of steroid hormone biosynthesis is cholesterol side chain cleavage reaction catalyzed by cytochrome P-450scc in mitochondria. Chogenital lipoid adrenal hyperplasia which is called as Prader Syndrome has been believed as a deficiency of cytochrome P-450scc. However, Yasuda, a collabolator of this research has reported that cDNA for cytochrome P-450scc of the patient is not different from that of normal subject, and cytochrome P-450scc protein is actually expressed in the patient testis. Miller and colleagues in US also reported that cDNAs for adrenodoxin reductase (AR) and adrenodoxin (AD) of the patient are not also different from those of normal subject as well as in the case of cDNA for cytochrome P-450scc. From these investigations, the reason why cholesterol side chain cleavage reaction is diminis … More hed in the patient with Prader Syndrome is not yet clear.In this study, we attempted to elucidate the mechanism of decrease in the activity being focused on the interactions among AR,AD,and cytochrome P-450scc using enzymological techniques. Blotting analyzes of AR,AD,and cytochrome P-450scc showed that the contents of these enzyme components in the testis of the patient were not so different from those of normal subject. Cholesterol side chain cleavage activity of the patient testis mitochondria was 35% of that of normal subject indicating that the activity of the patient testis mitochondria is not always zero level. Phospholipid/protein value of the patient testis mitochondria was 0.380 whereas the value of the normal subject was 0.659, indicating that phospholipid of the patient testis mitochondria decreased significantly. We extracted phospholipid from mitochondria of the patient testis and analyzed the content of subclass of phospholipids. We found that the content of lysolecithin in the patient testis mitochondria increased 7-fold of that of normal subject. Lysolecithin is known to inhibit cholesterol side chain cleavage activity in mitochondria. We also confirmed the inhibitory effect of lysolecithin in the reconstituted system using AR,AD,and cytochrome p-450scc purified from bovine adrenal cortex. These results suggest that the increased level of lysolecithin found in testis mitochondria from the patient is related to the mechanism of decrease in the activity in congenital lipoid adrenal hyperplasia. Less