ACTIVATION OF ACYL-COENZYME A-CHOLESTEROL ACYLTRANSFERASE BY CHOLESTEROL OR BY OXYSTEROL IN A CELL-FREE SYSTEM

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and ch olesterol to form cholesteryl esters. It is an integral membrane prote in located in the endoplasmic reticulum. Experiments performed in inta ct mammalian cells have shown that the rate of cholesteryl ester synth esis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL ) or oxygenated sterols such as 25-hydroxycholesterol to the growth me dium, However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., H uh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells, Uninfected Sf9 cells do not express det ectable ACAT-like activity. Infecting these cells with recombinant vir us containing ACAT cDNA caused these cells to express high levels of A CAT protein and high levels of ACAT activity when assayed in vitro. Th e catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells, The combinati on of high level of ACAT protein expression and the low level of cellu lar cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulati on of ACAT by sterols can be readily demonstrated, Using this system, we have shown that cholesterol itself can serve as an ACAT activator i n vitro, in addition to its role as an ACAT substrate. The current wor k provides the experimental basis to hypothesize that, inside mammalia n cells, cholesterol itself may serve as a physiological regulator of ACAT.