Recombinant acyl-CoA : cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesiclesin a highly cooperative manner

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-I, was identified in 1993 through an expression cloning approach, We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approxima tely 7000-fold from crude cell extracts by first solubilizing the cell memb ranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio ]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody af finity column and an immobilized metal affinity column. The final preparati on is enzymologically active and migrates as a single band at 54 kDa on SDS -polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelle s containing sodium taurocholate, phosphatidylcholine, and cholesterol rema ins catalytically active. The cholesterol substrate saturation curves of th e enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These re sults support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol.