FUNCTION OF ASCORBIC ACID IN STERIOD BIOSYNTHESIS

本提案的目标是确定 类固醇生物合成和线粒体中的抗坏血酸(AA) 能量代谢。NADH-SDA还原酶与AA电子 Mt类固醇的转运途径(NADH-AA ETP) 羟基酶将通过研究NADH的影响来确定， AA和肾上腺素还原酶特异性抗体及 EPR测定肾上腺素对其氧化还原状态的影响 细胞色素P450还原动力学的光谱研究 用双波长分光光度法测量，并在 用放射免疫法测定羟化活性和 反相高效液相色谱法。NADH/AA的能源需求 ETP支持的羟基化反应将通过研究 氧化磷酸化抑制剂对这一活性的影响。 NADH/AA ETP与Mt质子势的耦合 不同之处将根据上述因素的影响而定 NADH/AA ETP上的抑制剂支持羟化，在mt. 用Intra测量跨膜电位和pH梯度 产后86Rb的mT/额外mT分布 弱酸14C-DMO。对 依赖于能量的AA的mt传输也将由以下因素决定 测量~(14)C-AA累积的能量学。这个 NADH/AA ETP对类固醇生物合成的重要性将是 脂质体包埋处理完整细胞的研究 抗体这一途径的酶的抗体。这一点的存在 ETP在其他类固醇合成组织中的作用将被研究。最后， NADH/AA ETP将电子转移到 呼吸酶将通过研究氧气来确定 荧光素的摄取、极谱和ATP合成- 荧光素酶测定法。界定NADH/AA的职能 类固醇生物合成中的ETP和Mt能量学中的ETP将促进我们的 了解维生素C在哺乳动物体内的生化作用 新陈代谢，并可能为异常原因提供新的见解 类固醇生物合成。

The objectives of this proposal are to determine the function of ascorbic acid (AA) in steroid biosynthesis and mitochondrial (Mt) energy metabolism. The NADH-SDA reductase and AA electron transport pathway (NADH-AA ETP) to the Mt steroid hydroxylases will be determined by studying the effects of NADH, AA and specific antibodies to adrenodoxin reductase and adrenodoxin on the redox state of adrenodoxin measured by EPR spectroscopy, on the kinetics of reduction of cytochrome P450 measured by dual wavelength spectrophotometry and on the hydroxylation activity measured by radioimmunoassay and reversed phase HPLC. The energy requirements of the NADH/AA ETP supported hydroxylation will be determined by studying the effects of inhibitors of oxidative phosphorylation on this activity. Coupling of the NADH/AA ETP to a Mt protonic potential difference will be determined from the effects of the above inhibitors on NADH/AA ETP supported hydroxylation, and on Mt transmembrane potentials and pH gradients measured by the intra Mt/extra Mt distribution of 86Rb induced by valinomycin and of the weak acid 14C-DMO, respectively. The requirement for an energy dependent Mt transport of AA will also be determined by measuring the energetics of 14C-AA accumulation. The importance of this NADH/AA ETP for steroid biosynthesis will be studied in intact cells by treating them with liposomal entrapped antibodies to the enzymes of this pathway. The existence of this ETP in other steroid synthesizing tissues will be studied. Finally, the possibility that the NADH/AA ETP transfers electrons to the respiratory enzymes will be determined by studying oxygen uptake, polargraphically and ATP synthesis by a luciferin- luciferase assay. Delineation of the function of the NADH/AA ETP in steroid biosynthesis and in Mt energetics will advance our understanding of the biochemical role of vitamin C in mammalian metabolism and may provide new insights into causes of abnormal steroid biosynthesis.