Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid.
We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors; D002417:Cattle; D003315:Cornea / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000276:immunology / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D007672:Kidney Cortex / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000378:metabolism*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D011817:Rabbits
D001095:Arachidonic Acids; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D006419:Heme Oxygenase (Decyclizing); D003579:Cytochrome Reductases; D009251:NADPH-Ferrihemoprotein Reductase