Acyl-CoA:cholesterol acyltransferase in Chinese hamster ovary cells. Enzyme activity determined after reconstitution in phospholipid/cholesterol liposomes.
Acyl-CoA:cholesterol acyltransferase from Chinese hamster ovary (CHO) cells was solubilized by deoxycholate, and then reconstituted in phosphatidylcholine/cholesterol liposomes. This reconstituted activity was totally dependent upon the cholesterol content of the mixture and showed saturation for cholesterol. Analysis of the reconstituted enzyme on linear Ficoll gradients shows that the enzyme has been incorporated into phospholipid/cholesterol liposomes. The CHO cell enzyme activity as measured by conventional assay (using cellular cholesterol as the substrate) was activated approximately 20-fold by low density lipoprotein. This activation process was independent of protein synthesis. When the above cell homogenates were assayed after optimal reconstitution, the activation produced by low density lipoprotein was essentially completely abolished. There was also no change in enzyme activity measured after reconstitution when cells were switched from sterol-containing medium to sterol-free medium, in contrast to a more than 7-fold drop in enzyme activity when assayed without reconstitution. These results suggest that the enzyme activity in intact cells is controlled by the content and composition of cellular lipids associated with the enzyme molecule. Since the intracellular messenger of low density lipoprotein is known to be cholesterol, it is likely that this enzyme activity in intact cells is primarily controlled by the cholesterol content in the vicinity of the enzyme molecule.
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002784:Cholesterol; D006224:Cricetinae; D003412:Cricetulus; D003840:Deoxycholic Acid / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008081:Liposomes; D010053:Ovary; D010713:Phosphatidylcholines; D002785:Sterol O-Acyltransferase / Q000378:metabolism*
D008081:Liposomes; D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; D002784:Cholesterol; D000217:Acyltransferases; D002785:Sterol O-Acyltransferase