The regulation of neutral cholesterol ester hydrolase activity by chan
ges in its phosphorylation state was studied in rat liver microsomes.
Treatment with cAMP-dependent protein kinase resulted in increased enz
yme activity, which was further enhanced by the addition of cAMP and M
gATP. Consistent activations were also achieved with MgCl2 and MgATP,
the magnesium effect being abolished by ethylenediaminetetraacetic aci
d and adenosine triphosphate. Cholesterol ester hydrolase was activate
d twofold by free calcium and Ca2+/calmodulin; this latter effect was
blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,
N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine.
The phosphatase inhibitors pyrophosphate and glycerophosphate led to m
arked and dose-dependent increases in esterase activity, whereas okada
ic acid elicited no effect. Further more, pyrophosphate and okadaic ac
id did not change the increases in enzyme activity promoted by Ca2+, C
a2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inacti
vated in a concentration-dependent manner by nonspecific alkaline phos
phatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-ac
tivated microsomes, a time-dependent loss of activation in cholesteryl
oleate hydrolysis was caused by alkaline phosphatase. These findings
suggest that microsomal cholesterol ester hydrolase is activated throu
gh cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivati
on is dependent on phosphatase action.