Purification and Control of Bovine Adrenal Cortical Cholesterol Ester Hydrolase and Evidence for the Activation of the Enzyme by a Phosphorylation

Abstract

A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 ×g supernatant is described. Preincubation of a crude enzyme extract with [γ-³²P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 ± 280 only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [α-³²P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 ×gsupernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and magnesium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ions was accompanied by a loss of ³²P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.