Studies on Adrenal Steroid Hydroxylases

Abstract

Abstract Adrenodoxin reductase and adrenodoxin have been shown to form a complex with a stoichiometric molar ratio of 1:1. The dissociation constant of the complex is found to be approximately 10-9 m in a low ionic strength medium, and approximately 10-6 m in a high ionic strength medium. The reductase is relatively labile upon heat treatment; the adrenodoxin-dependent cytochrome c reductase activity decreases about 80% when reductase is treated at 45° for 15 min. The presence of adrenodoxin largely protects the inactivation. The NADPH-2,6-dichloroindophenol reductase activity of adrenodoxin reductase is stimulated either by the addition of adrenodoxin or by increasing the concentrations of salts. Antiserum prepared against adrenodoxin has no effect on the adrenodoxin-stimulated NADPH-2,6-dichloroindophenol reductase activity. The NADPH-cytochrome c reductase activity of the complex has a maximal activity at an ionic strength of 0.04, and no activity at an ionic strength over 0.4. Antiserum against adrenodoxin completely inhibits this activity. The reductase-adrenodoxin complex has been shown to produce superoxide anion radicals in the presence of NADPH and molecular oxygen. The site responsible for superoxide anion radical generation is predominantly on the iron-sulfur protein. Adrenodoxin reductase, adrenodoxin, or their complex fails to show any indication of a stable stoichiometric complex with partially purified cytochrome P-450, as judged by a Sephadex gel filtration technique.