Human acyl-CoA : cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro

Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cho lesterol homeostasis and in atherosclerosis. ACAT-1 may function as an allo steric enzyme, We took a multifaceted approach to investigate the subunit c omposition of ACAT-1, When ACAT-1 with two different tags were co-expressed in the same Chinese hamster ovary cells, antibody specific to one tag caus ed co-immunoprecipitation of both types of ACAT-1 proteins. Radioimmunoprec ipitations of cells expressing the untagged ACAT-1 or the 6-histidine-tagge d ACAT-1 yielded a single radiolabeled band of predicted size on SDS-polyac rylamide gel electrophoresis. These results show that ACAT-1 exists as homo -oligomers in intact Chinese hamster ovary cells. We solubilized HisACAT-1 with the detergent deoxycholate or CHAPS (3-[(3-cholamidopropyl)-dimethylam moniol]-1-propane-sulfonic acid), performed gel filtration chromatography a nd sucrose density gradient centrifugations in H2O and D2O, and determined the Stokes radii and sedimentation coefficients of the HisACAT1-detergent c omplexes, The estimated molecular mass of HisACAT-1 is 263 kDa, which is 4 times that of the HisACAT-1 monomer (69 kDa), Finally, cross-linking experi ments in intact cells and in vitro show that the increase in cross-linker c oncentrations causes an increase in size of the HisACAT-1-positive signals, forming material(s) 4 times the size of the monomer, supporting the conclu sion that ACAT-I is a homotetrameric enzyme.