大鼠肝脏微粒体二酰基甘油酰基转移酶的增溶、部分纯化及特性鉴定

Solubilization, partial purification and characterization of rat liver microsomal diacylglycerol acyltransferase.

作者信息

Polokoff M A, Bell R M

出版信息

Biochim Biophys Acta. 1980 Apr 18;618(1):129-42. doi: 10.1016/0005-2760(80)90060-0.

Abstract

The diacylglycerol acyltransferase (EC 2.3.1.20) activity of rat liver microsomes was solubilized with deoxycholate, cholate, and Triton X-100. The cholate-solubilized activity proved to be more stable than the deoxycholate-solubilized activity. Cholate caused less interference with the diacylglycerol acyltransferase assay than Triton X-100. The cholate-solubilized activity was purified 3- to 4-fold by Sepharose 4B chromatography, and could be separated from most of the cholate-solubilized protein by centrifugation in a 10--20% sucrose gradient. The purification led to an overall purification of 9-fold with a recovery of 80%. The gradient fractions were analyzed for protein, RNA, and phospholipid content as well as for several enzyme activities of glycerolipid biosynthesis. The partially purified fractions were delipidated and activity was stimulated 5-fold by the addition of sonicated microsomal phospholipids. The soluble, gradient-puridied diacylglycerol acyltransferase activity was strongly dependent on added magnesium.

摘要

用脱氧胆酸盐、胆酸盐和 Triton X - 100 溶解大鼠肝脏微粒体的二酰基甘油酰基转移酶（EC 2.3.1.20）活性。结果证明，胆酸盐溶解的活性比脱氧胆酸盐溶解的活性更稳定。与 Triton X - 100 相比，胆酸盐对二酰基甘油酰基转移酶测定的干扰更小。通过琼脂糖 4B 层析将胆酸盐溶解的活性纯化了 3 至 4 倍，并且通过在 10 - 20%蔗糖梯度中离心可将其与大部分胆酸盐溶解的蛋白质分离。该纯化过程实现了 9 倍的总纯化，回收率为 80%。对梯度级分进行蛋白质、RNA、磷脂含量分析以及甘油脂质生物合成的几种酶活性分析。对部分纯化的级分进行脱脂处理，并通过添加超声处理的微粒体磷脂使活性提高了 5 倍。可溶性、梯度纯化的二酰基甘油酰基转移酶活性强烈依赖于添加的镁。