Characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human adrenal cortex.
Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.
D000302:Adrenal Cortex / Q000201:enzymology*; D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000311:Adrenal Glands / Q000032:analysis; D002784:Cholesterol / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007163:Immunosorbent Techniques; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D009249:NADP / Q000378:metabolism; D010765:Phosphorylase Phosphatase / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology
D009249:NADP; D012969:Sodium Fluoride; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D010765:Phosphorylase Phosphatase; D004229:Dithiothreitol