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

胆固醇侧链裂解反应及其相关遗传性疾病的机制分析

• 批准号: 05680565
• 负责人: HARA Takayuki
• 金额: $ 1.34万
• 依托单位: NAKAMURA GAKUEN UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680565/
• 关键词: STEROID HORMONE; HEREDITARY DISEASE; CHOLESTEROL SIDE CHAIN CLEAVAGE REACTION; CYTOCHROME P-450scc; CONGENITAL LIPOID ADRENAL HYPERPLASIA; PHOSPHOLIPID; LYSOLECITHIN; MITOCHONDRIA; コレステロール; アドレノドキシン還元酵素; アドレノドキシン; 副腎リポイド過形成症; 反応メカニズム; 遺伝病解析

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

甾体激素在高等动物的正常生命活动中起着重要的作用，这些激素的紊乱会导致动物的严重疾病。甾体激素生物合成的限速步骤是线粒体中细胞色素P-450 SCC催化的胆固醇侧链裂解反应。Chogential lipoid adrenal hyperplasia（ChG）又称Prader综合征，被认为是细胞色素P-450 SCC缺乏所致。然而，本研究的合作者Yasuda报道，患者的细胞色素P-450 SCC的cDNA与正常受试者的cDNA没有不同，并且细胞色素P-450 SCC蛋白实际上在患者睾丸中表达。美国米勒等也报道了该患者的肾上腺氧还蛋白还原酶（AR）和肾上腺氧还蛋白（AD）的cDNA与正常人的cDNA以及细胞色素P-450 SCC的cDNA没有差异。从这些调查中，胆固醇侧链裂解反应的原因是亚氨基 ...更多信息 在本研究中，我们试图通过酶学技术阐明AR、AD和细胞色素P-450 SCC之间相互作用的机制。AR、AD和细胞色素P-450 SCC的印迹分析表明，患者睾丸中这些酶组分的含量与正常人无明显差异。患者睾丸线粒体胆固醇侧链裂解活性为正常人的35%，表明患者睾丸线粒体的活性并不总是零水平。患者睾丸线粒体的磷脂/蛋白值为0.380，而正常人的值为0.659，表明患者睾丸线粒体的磷脂显著减少。从患者睾丸线粒体中提取磷脂，分析磷脂亚类的含量。结果发现，病人睾丸线粒体中溶血素含量比正常人高7倍。已知溶血素抑制线粒体中的胆固醇侧链裂解活性。我们还证实了在使用AR，AD和从牛肾上腺皮质纯化的细胞色素p-450 SCC的重建系统中的溶血素的抑制作用。这些结果表明，从病人睾丸线粒体中发现的溶血素水平的增加与先天性类脂质肾上腺皮质增生症的活性降低的机制有关。少

项目成果

Y.Sakai, T.Yanase, Y.Okabe, T.Hara, M.R.Waterman R.Takayanagi, M.Haji, and H.Nawata: ""No Mutation in Cytochrome P-450 Side Chain Cleavage in a Patient with Congenital Lipoid Adrenal Hyperplasia"" J.Clin.Endocrinol.Metab.79. 1198-1201 (1994)

Y.Sakai、T.Yanase、Y.Okabe、T.Hara、M.R.Waterman R.Takayanagi、M.Haji 和 H.Nawata：“先天性类脂肾上腺患者细胞色素 P-450 侧链断裂无突变

S.Watabe,T.Hara,et al.: "In Vitro Degradation of Mitochondrial Proteins by ATP-Dependent Protease in Bovine Adrenal Cortex" J.Biochem.113. 672-676 (1993)

S.Watabe、T.Hara 等人：“牛肾上腺皮质中 ATP 依赖性蛋白酶对线粒体蛋白的体外降解”J.Biochem.113。

T.Hara, and M.Takeshima: ""Conclusive Evidence of a Quaternary Cluster Model for Cholesterol Side Chain Cleavage Reaction Catalyzed by Cytochrome P-450scc"" In Cytochrome P-450, Biochemistry, Biophysics, and Molecular Biology (M.C.Lechnered.) pp.417-420,

T.Hara 和 M.Takeshima：“细胞色素 P-450scc 催化的胆固醇侧链裂解反应的四元簇模型的结论性证据”，《细胞色素 P-450、生物化学、生物物理学和分子生物学》(M.C.Lechnered.)

Takayuki Hara: "Conclusive Evidence of a Quaternary Cluster Model for Cholesterol Side Chain Cleavage Reaction Catalyzed by Cytochrome P-450scc" In Cytochrpme P-450 John Libbey Eurotext. 417-420(916) (1994)

Takayuki Hara：“细胞色素 P-450scc 催化的胆固醇侧链裂解反应的四元簇模型的结论性证据”，载于 Cytochrpme P-450 John Libbey Eurotext。

Yoshiyuki Sakai: "No Mutation in Cytochrome P-450 Side Chain Cleavage in a Patient with Congenital Lipoid Adrenal Hyperplasia" J. Clin. Endocrinol. Metab.79. 1198-1201 (1994)

Yoshiyuki Sakai：“先天性类脂性肾上腺增生患者的细胞色素 P-450 侧链裂解没有突变”J. Clin。