The activation of microsomal steroid 21-hydroxylase by cytosol from the cortex of bovine adrenals.

At low microsome concentrations, the addition of cytosol from bovine adrenal cortex markedly accelerates the rate of hydroxylation of 17-hydroxyprogesterone at C-21. The detection of this effect was made possible by the development of a new, rapid, and sensitive procedure for the measurement of the initial rate kinetics of steroid 21-hydroxylase. This procedure is based on the fact that the reactant, 17-hydroxyprogesterone, possesses solvent partition properties which are different from those of the product, cortexolone. The specificity of the assay was confirmed by the isolation of only one product which was identified as cortexolone by radiochemical techniques. This assay procedure has great sensitivity and makes possible the accurate determination of the Michaelis constant at low enzyme concentrations. The Km for 17-hydroxyprogesterone with saturating amounts of TPNH was found to be 0.3 micronM. At the low microsome concentrations permitted by this assay, the addition of cytosol has a profound effect upon the rate of hydroxylation. The rate is markedly accelerated although the Km for the substrate is not altered. Neither the substrate nor the carbon monoxide-induced difference spectra are changed by the addition of cytosol, suggesting that activation by cytosol dose not affect the catalytic unit of the 21-hydroxylase complex.