The function of NADH-semidehydroascorbate reductase and ascorbic acid in corticosteroid hydroxylation.
We have demonstrated in rat adrenal (Natarajan, R.D. and Harding, B.W. (1985) J. Biol. Chem. 260, 3902-3905) that NADH-semidehydroascorbate reductase and ascorbate participate in an electron transport pathway (ETP) supplying reducing equivalents from NADH to cytochrome P-450scc. Here, we demonstrate that this ascorbate dependent ETP also supplies reducing equivalents to cytochrome P-450(11 beta/18) in both rat adrenal and bovine adrenal cortex. The activity is dependent upon addition of catalase or upon 'cold shock' treatment of isolated mitochondria. Comparison of the rates of 11 beta- and 18-hydroxylation supported by this ETP and by the classical pathway supported by various TCA cycle intermediates suggests that in vivo the ascorbate dependent pathway may be essential for maximal flow of reducing equivalents to the mitochondrial hydroxylases. Partial reconstitution of the ascorbate dependent 11 beta/18-hydroxylase activity was achieved with purified bovine outer mitochondrial and inner mitochondrial membranes fortified with supernatant from sonified mitochondria all preincubated with phosphatidyl choline. These preparations no longer require catalase or 'cold shock' treatment. Ascorbate and NADH-semidehydroascorbate reductase are unable to support 17 alpha- or 21-hydroxylase activity in isolated bovine adrenal cortical microsomes whether incubated with purified outer mitochondrial membranes or not.
Natarajan
R D
RD
Department of Medicine, LAC/USC Medical Center, Los Angeles 90033.
Harding
B W
BW
D000302:Adrenal Cortex / Q000201:enzymology; D000305:Adrenal Cortex Hormones / Q000378:metabolism; D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000378:metabolism*
D000305:Adrenal Cortex Hormones; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D001205:Ascorbic Acid