Balasubramaniam S, Venkatesan S, Mitropoulos K A, Peters T J
Biochem J. 1978 Sep 15;174(3):863-72. doi: 10.1042/bj1740863.
To determine the submicrosomal distribution of acyl-CoA-cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA-cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA-cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [(14)C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7alpha-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7alpha-hydroxylase and the other acting as substrate for acyl-CoA-cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.
为了确定酰基辅酶A - 胆固醇酰基转移酶和胆固醇酯在亚微粒体中的分布，对大鼠肝脏的微粒体部分和经洋地黄皂苷处理的微粒体制剂进行了蔗糖密度梯度分析离心。对于未处理的微粒体部分，酰基辅酶A - 胆固醇酰基转移酶的分布图谱和中位密度与RNA的非常相似。这与羟甲基戊二酰辅酶A还原酶和胆固醇7α - 羟化酶形成对比，后者局限于核糖体包被低的内质网内膜。在经洋地黄皂苷处理的微粒体制剂中，未检测到酰基辅酶A - 胆固醇酰基转移酶的活性。用微量的[(14)C]胆固醇标记未处理的微粒体部分，然后对标记的微粒体部分进行亚分级分离，结果表明，各个亚分级分离物在体外获得的胆固醇酯的比放射性在所有亚分级分离物中相似，但与同一亚分级分离物在体外获得的7α - 羟基胆固醇的比放射性不同。这些结果证明在内质网的膜中存在两池胆固醇，一池作为胆固醇7α - 羟化酶的底物，另一池作为酰基辅酶A - 胆固醇酰基转移酶的底物。未处理和经洋地黄皂苷处理的微粒体部分中存在的胆固醇酯的主要部分分布在与光滑内质网的膜相似的密度处，且密度低于高尔基体光滑膜的密度。未处理和经洋地黄皂苷处理的微粒体部分的亚分级分离物中未酯化胆固醇与酯化胆固醇的浓度比在质膜的最大分布处最高。
