Chasalow F I, Lieberman S
J Biol Chem. 1979 May 25;254(10):3777-81.
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.
在微粒体浓度较低时，添加来自牛肾上腺皮质的胞液能显著加快17 - 羟孕酮在C - 21位的羟化速率。一种用于测定类固醇21 - 羟化酶初始速率动力学的新的快速灵敏方法的开发，使得这种效应的检测成为可能。该方法基于反应物17 - 羟孕酮具有与产物皮质醇不同的溶剂分配特性这一事实。通过仅分离出一种经放射化学技术鉴定为皮质醇的产物，证实了该测定方法的特异性。这种测定方法具有很高的灵敏度，使得在低酶浓度下准确测定米氏常数成为可能。发现对于17 - 羟孕酮，在TPNH饱和量时的Km为0.3微摩尔。在该测定所允许的低微粒体浓度下，添加胞液对羟化速率有深远影响。尽管底物的Km未改变，但速率显著加快。添加胞液既不改变底物，也不改变一氧化碳诱导的差异光谱，这表明胞液的激活并不影响21 - 羟化酶复合物的催化单元。
