Goodwin C D, Margolis S
J Lipid Res. 1976 May;17(3):297-303.
Two new methods are described for the study of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. (1) Endoplasmic reticulum was rapidly prepared by diluting a 10,000 g supernatant with buffer containing 8 mM calcium chloride. The yield of protein and the specific activity of HMG CoA reductase in the pellet subsequently obtained by low speed centrifugation were nearly identical to those in the microsomal pellet prepared by ultracentrifugation. This technique may be particularly useful in studies of the rapid, in vitro modulation of the enzyme. (2) Mevalonolactone was extracted into benzene from the HMG CoA reductase assay mixture with an efficiency of 58%. There was less than 1% extraction of HMG CoA, acetoacetate, or beta-hydroxybutyrate. The extracted mevalonolactone was at least 98% pure as judged by thin-layer chromatography with four different solvent systems. These improved methods should significantly aid studies of the physiological importance of HMG CoA reductase.
本文描述了两种用于研究肝脏3-羟基-3-甲基戊二酰辅酶A（HMG CoA）还原酶的新方法。（1）通过用含有8 mM氯化钙的缓冲液稀释10,000 g上清液来快速制备内质网。随后通过低速离心获得的沉淀中蛋白质产量和HMG CoA还原酶的比活性与通过超速离心制备的微粒体沉淀中的几乎相同。该技术在酶的快速体外调节研究中可能特别有用。（2）甲羟戊酸内酯从HMG CoA还原酶测定混合物中以58%的效率萃取到苯中。HMG CoA、乙酰乙酸或β-羟基丁酸的萃取率低于1%。通过用四种不同溶剂系统进行薄层色谱分析判断，萃取的甲羟戊酸内酯纯度至少为98%。这些改进的方法应能显著有助于对HMG CoA还原酶生理重要性的研究。
