The submicrosomal localization of acyl-coenzyme A-cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver.
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.
D000214:Acyl Coenzyme A; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002500:Centrifugation, Isopycnic; D002784:Cholesterol / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D006888:Hydroxycholesterols / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D002785:Sterol O-Acyltransferase / Q000378:metabolism*
D000214:Acyl Coenzyme A; D002788:Cholesterol Esters; D006888:Hydroxycholesterols; D002784:Cholesterol; D002790:Cholesterol 7-alpha-Hydroxylase; D000217:Acyltransferases; D002785:Sterol O-Acyltransferase; D004072:Digitonin