Lehoux J G, Kandalaft N, Belisle S, Bellabarba D
Endocrinology. 1985 Oct;117(4):1462-8. doi: 10.1210/endo-117-4-1462.
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.
我们的研究比较了来自人类转移性男性化癌的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶与来自肾脏供体的正常肾上腺的该还原酶的特性。当使用含有5 mM乙二胺四乙酸（EDTA）、250 mM氯化钠、1 mM苯甲基磺酰氟、0.1 mM亮抑酶肽和5.5 mM二硫苏糖醇（DTT）的50 mM咪唑缓冲液（pH 7.2）时，获得了酶测定的最佳条件。在添加底物之前进行30分钟的预温育期可使还原酶活性提高1.75倍。在粗微粒体制剂中，肿瘤组织和正常组织的米氏常数（Km）值相似，在4至5 microM（S）HMG-CoA之间变化。在匀浆和孵育介质中存在氟化钠会降低最大反应速度，但不会影响Km。部分纯化的大鼠肝脏磷酸化酶磷酸酶制剂或来自癌组织的类似制剂可将在氟化钠存在下制备的微粒体的还原酶活性恢复到最高水平。在癌组织微粒体制剂中发现NADP的Km为96 microM。在单硫甘油或DTT存在下对微粒体进行预温育会导致还原酶活性增加，这表明可能存在由二硫键连接的单元组成的无活性酶前体。在存在增加量的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的情况下孵育的DTT激活酶的反应显示出S形动力学。在还原条件下，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹显示存在一条92.5K分子量的蛋白带，该带与大鼠抗还原酶抗体发生反应。癌组织不同区域的还原酶活性在679 - 1763 pmol/mg蛋白之间变化，平均为1146 pmol。在三个正常肾上腺中，我们测得的值分别为23.4、48.1和36 pmol。我们得出结论，HMG-CoA还原酶活性在人类肾上腺癌中表达升高。
