Abstract
Acyl-CoA:cholesterol acyltransferase was found predominantly (85%) in RNA-rich microsomes, the rest being in RNA-poor and smooth microsomes. However, the esterified cholesterol concentration of smooth microsomes was 2-fold greater than that of RNA-rich microsomes, suggesting the possibility of an interaction between RNA-rich and smooth microsomes. The distribution of cholesteryl ester between microsome subfractions was examined after incubation of a mixture of RNA-rich and smooth microsomes with [1-14C]palmitoyl-CoA. Based upon specific acyl-CoA:cholesterol acyltransferase activities of the individual fractions, only 31 +/- 3% of the total cholesteryl ester radioactivity should have been found in the smooth component. However, the smooth microsomes contained 54 +/- 3% (p < 0.01) of the radioactive cholesteryl esters. The entrapment of radioactive cholesteryl ester in the smooth microsomes could not be accounted for by passive transfer of cholesteryl ester from RNA-rich microsomes to smooth microsomes. It is proposed that cholesterol in the smooth microsomal membranes may have been esterified by acyl-CoA:cholesterol acyltrasferase located on the surface of RNA-rich microsomes with the resulting cholesteryl ester retained in the smooth microsomes. This hypothesis was strengthened by the observation that acyl-CoA:cholesterol acyl-transferase was located on the cytoplasmic surface of the RNA-rich microsomal vesicle.
Highlights
From the *Research Service, Wadsworth Veterans Administration Hospital Center, Los Angeles, California 90073, and the SDiuision ofCardiology, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024
In preliminary experiments we found that thesmooth com- dient: 3.6 ml of the 10,OOO X g supernatant containing 30 mgof ponent of rat liver microsomes was enriched with newly synthesized cholesteryl esters more than could be accounted for by the cholesteryl-esterifying activity of the smooth microsomes
RNA-rich and smooth zones were removed from the gradient, and washed by suspension and centrifugation, and the pellets assayed for cholesteryl ester radioactivity
Summary
Microsomal Cholesteryl Ester (60°C for 5 min) RNA-rich microsomes; fresh or heated smooth microsomes; and all possible combinations of the fresh and heated preparations (Tables IV and V). RNA-rich and smooth zones were removed from the gradient, and washed by suspension and centrifugation, and the pellets assayed for cholesteryl ester radioactivity. After incubation for 16 min the reaction mixture was cooled to 4’C; added directly (without heating) to a nonradiactive, heat-activated microsomal pool (30 mg of protein); and the mixture was resolved into components by centrifugation in the two sucrose gradients described above. RNA-rich microsomes (50 mg of protein) were suspended in 2 ml of 0.25 M sucrose 50 mM Tris buffer (pH 7.5) and incubated at 30°C for 10 min with trypsin (5 mg) and pronase (5 mg). NADH-cytochrome c reductase [4], and glucose-6-phosphatase [9] by referenced methods
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