Recombinant acyl-CoA : cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesiclesin a highly cooperative manner
Ccy. Chang et al., Recombinant acyl-CoA : cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesiclesin a highly cooperative manner, J BIOL CHEM, 273(52), 1998, pp. 35132-35141
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.