Regulation of triacylglycerol synthesis in the liver. Modulation of diacylglycerol acyltransferase activity in vitro.

Acyl-CoA:1,2-diacylglycerol O-acyltransferase (EC 2.3.1.20) of rat liver microsomes could be inactivated in vitro by incubation with ATP, Mg2e , and a 105,000 x g rat liver supernatant. Of the nucleotides tested, ATP was the most effective in inactivating diacylglycerol acyltransferase. The rate of this inactivation was not influenced by the addition of cAMP. Treatment of rat liver microsomes with high concentrations of the catalytic subunit of cAMP-dependent protein kinase did not result in the inactivation of diacylglycerol acyltransferase. The activity of diacylglycerol acyltransferase was lower in microsomes isolated in the presence of fluoride (50 mM) than in control microsomes isolated from homogenates containing chloride (50 mM) instead of fluoride. Interestingly, the activity of diacylglycerol acyltransferase in microsomes isolated in the presence of fluoride and that in control microsomes were both reduced to the same level upon treatment of the microsomes with Mg2 +, ATP. and 105,000 x g supernatant. In order to investigate whether the inactivated diacylglycerol acyltransferase could be reactivated the microsomes were reisolated, washed, and subsequently incubated with 105,000 x g supernatant of rat liver. Microsomal diacylglycerol acyltransferase could indeed be reactivated by a factor present in the 105,000 x g supernatant. The activating factor appeared to be heat-labile, nondialyzable, and trypsin-sensitive. The reactivation of microsomal diacylglycerol acyltransferase was inhibited in the presence of fluoride (50 mM). Both inactivation and reactivation of diacylglycerol acyltransferase appeared to be reversible processes; reactivated microsomal diacylglycerol acyltransferase could be inactivated again by incubation with Mg2 +, ATP, and 105,000 x g supernatant and subsequently reactivated again in the presence of 105,000 x g supernatant. These findings are consistent with a model in which microsomal diacylglycerol acyltransferase is interconvertible between catalytically inactive and active states, possibly via a phosphorylation-dephosphorylation mechanism.