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.
