Solubilization, partial purification, and reconstitution in phosphatidylcholine-cholesterol liposomes of acyl-CoA:cholesterol acyltransferase.
Acyl-CoA:cholesterol acyltransferase (ACAT) was solubilized from pig liver microsomes with a combination of 1:1% deoxycholate and 1 M potassium chloride. This solubilized activity was then reconstituted in lipid vesicles by diluting the extract into a solution of phosphatidylcholine, cholesterol, and sodium cholate, followed by dialysis. The reconstituted activity was shown to be dependent upon cholesterol in the reconstitution mixture and also shown to vary with changes in the phospholipid headgroup: phosphatidylethanolamine was most active, phosphatidylcholine was next, and phosphatidylserine or phosphatidylinositol was inhibitory. The reconstituted activity showed a migration pattern of ficoll gradients that was distinct from that of the unreconstituted enzyme and similar to that of phospholipid-cholesterol liposomes. These method provide a technique to assay the ACAT activity in defined lipid environment. The solubilized ACAT fraction was further purified by ammonium acetate fractionation and Sepharose 4B column chromatography. The entire purification procedure yielded a 150-fold increase in ACAT specific activity with 40% of the original activity recovered.
D000217:Acyltransferases / Q000302:isolation & purification*; D000818:Animals; D002784:Cholesterol; D066298:In Vitro Techniques; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology; D010713:Phosphatidylcholines; D012995:Solubility; D002785:Sterol O-Acyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
D008081:Liposomes; D010713:Phosphatidylcholines; D002784:Cholesterol; D000217:Acyltransferases; D002785:Sterol O-Acyltransferase