EFFECT OF EXOGENOUS CHOLESTEROL AND DITHIOTHREITOL ON THE ACTIVITY OFHUMAN LIVER MICROSOMAL ACYL-COENZYME A-CHOLESTEROL ACYLTRANSFERASE (ACAT)

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is the intracellula r enzyme responsible for the esterification of cholesterol with long-c hain fatty acyl-CoA derivatives and has been implicated in atheroscler osis and gallstone disease. The effects of exogenous cholesterol and d ithiothreitol (DTT) on the ACAT activity of human liver microsomes hav e been determined. Pre-incubation of microsomes with exogenous cholest erol gave a marked stimulation of activity. Experiments with [H-3]chol esterol and [C-14]oleoyl-CoA indicated the time course of equilibratio n of exogenous with endogenous cholesterol as ACAT substrates, and sho wed that ACAT activity could be accurately measured using [H-3]cholest erol/Tween 80, providing that the concentration of endogenous microsom al cholesterol was also determined. Pre-incubation of liver microsomes for 90 min in the presence of 2 mmol/l DTT and. exogenous cholesterol /Tween 80 resulted in a 60% reduction in ACAT activity, compared with the corresponding activity when DTT was omitted. If microsomes were pr e-incubated with DTT prior to the pre-incubation with; exogenous chole sterol/Tween 80, an 85-90% reduction in ACAT activity occurred. In con trast, pre-incubation of microsomes with DTT in the absence of exogeno us cholesterol/Tween 80 (only endogenous cholesterol present) resulted , initially in a stimulation of ACAT activity; on further pre-incubati on, activity returned to control levels. These results indicate that t he supply of cholesterol to the enzyme active site is an important fac tor in ACAT assays in vitro and that DTT has a major effect on this pr ocess, suggesting that these factors may be important in controlling A CAT activity in vivo.