Enzymic studies on adrenocortical deoxycorticosterone 11beta-hydroxylase system.

Radiometric methods for the assay of deoxycorticosterone 11beta-hydroxylase and for the determination of NADP on a microscale were developed. The determination of NADP was based on the quantitative conversion of 6-phospho[1-14C]gluconate to 14CO2 by the action of 6-phosphogluconate dehydrogenase. Using these methods NADPH oxidase activity of the adrenodoxin reductase-adrenodoxin system as well as kinetic properties of deoxycorticosterone 11beta-hydroxylase (cytochrome P-450) were investigated. The NADPH oxidase activity observed in the presence of adrenodoxin reductase, adrenodoxin, and O2, but in the absence of cytochrome P-450 and deoxycorticosterone, were functions of O2 and adrenodoxin concentrations and represented the autooxidation of reduced adrenodoxin which resulted in the production of H2O2. Due to the rapid autooxidizability of reduced adrenodoxin, only a small fraction of electrons conveyed from NADPH to adrenodoxin by way of adrenodoxin reductase was utilized for the deoxycorticosterone 11beta-hydroxylase reaction under the conditions employed.