title: Some characteristics of human adrenal microsomal 21-hydroxylase activity.
abstract: The following general characteristics of 21-hydroxylase activity were determined using pooled microsomes obtained from three glands. Enzyme activity exhibited a broad pH dependence, being optimal between pH 7.4-pH 7.8, and was maximal with NADPH in the range 2 to 4.75 X 10(-4)mol/l. No microsomal 21-hydroxylase activity was detected in the absence of NADPH or substrate and when heat denatured microsomes were employed. Enzyme activity was depressed by greater than 75% in the presence of 100% oxygen or nitrogen. In a second set of experiments, microsomal fractions were prepared individually from 7 glands. In the presence of 17 alpha-hydroxy progesterone (2.0 X 10(-7) and 2.0 X 10(-6)mol/l) product formation was linear with time for up to 90 s when the microsomal protein concentration was 5, 10 and 20 micrograms/ml. Between 5 and 30% of the substrate was converted during the first 60 s. In 5/7 of the glands the addition of the autologous cytosol (20 micrograms protein/ml) was without effect, and enzyme activity (using a 60 s reaction and either 2.0 X 10(-7) or 2 X 10(-6)mol/l 17 alpha-hydroxy progesterone was directly proportional to the microsomal protein concentration (range 0-20 micrograms/ml). With the other 2 adrenals 21-hydroxylation was not proportional to the same range of microsomal protein concentrations, although it became so upon the addition of cytosol, which significantly augmented activity. There was considerable variation in enzyme activity between glands from different individuals (Vmax ranging from 2.6 to 16.6 X 10(-9) mol/min/mg protein) and in the apparent Km's (from 0.22 to 1.1 X 10(-6)mol/l). In the two preparations sensitive to cytosol, the Vmax increased 2-fold, and the Km was 3 times lower. Cytosol was without effect upon the kinetic characteristics of the other 5 microsomal preparations. Ascorbic acid (1 X 10(-3) mol/l) depressed enzyme activity by 25-43% whereas oxidised and reduced glutathione (1 X 10(-3) mol/l) showed a slight and variable effect upon 21-hydroxylation.
mesh_terms: D019326:17-alpha-Hydroxyprogesterone; D000311:Adrenal Glands / Q000201:enzymology*; D001205:Ascorbic Acid / Q000494:pharmacology; D003350:Cortodoxone / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006908:Hydroxyprogesterones / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D013255:Steroid 21-Hydroxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*
chemical_list: D006908:Hydroxyprogesterones; D009249:NADP; D019326:17-alpha-Hydroxyprogesterone; D013250:Steroid Hydroxylases; D013255:Steroid 21-Hydroxylase; D005978:Glutathione; D001205:Ascorbic Acid; D003350:Cortodoxone