FUNCTION OF ASCORBIC ACID IN STERIOD BIOSYNTHESIS

本提案的目的是确定 抗坏血酸（AA）在类固醇生物合成和线粒体（Mt） 能量代谢 NADH-SDA还原酶与AA电子 Mt类固醇的NADH-AA ETP转运途径 羟化酶将通过研究NADH的作用来确定， AA和肾上腺氧还蛋白还原酶的特异性抗体， 肾上腺素对肾上腺素氧化还原状态的影响 光谱法，对细胞色素P450的还原动力学 通过双波长分光光度法测量， 通过放射免疫测定法测量羟基化活性， 反相HPLC。 NADH/AA的能量需求 ETP支持的羟基化将通过研究 氧化磷酸化抑制剂对此活性的影响。 NADH/AA ETP与Mt质子势的耦合 差异将根据上述影响确定 抑制剂对NADH/AA ETP支持的羟基化，并对MT 跨膜电位和pH梯度测量的内 缬氨霉素诱导的86 Rb的Mt/extra Mt分布 弱酸14 C-DMO。 之规定 AA的能量依赖性MT运输也将由以下因素决定： 测量14 C-AA积累的能量学。 的 这种NADH/AA ETP对类固醇生物合成重要性将是 在完整的细胞中研究， 抗体的酶的途径。 存在这种 将研究其他类固醇合成组织中的ETP。 最后， NADH/AA ETP将电子转移到 呼吸酶将通过研究氧气 吸收，极谱和ATP合成的一个Escherin- 荧光素酶测定。 说明NADH/AA的功能 ETP在类固醇生物合成和MT能量学将促进我们的 了解维生素C在哺乳动物中的生物化学作用 代谢，并可能提供新的见解异常的原因， 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.