Hormone-sensitive cholesterol ester hydrolase of bovine adrenal cortex: Identification of the enzyme protein

Abstract

Cholesterol ester hydrolase catalyses one of the rate-limiting steps in steroidogenesis in the adrenal cortex, releasing cholesterol from the stores of cholesterol esters in the cytoplasm [ 11. The activity of the enzyme is elevated in response to administration of adrenocorticotropic hormone (ACTH) and under conditions of stress, which lead to increased steroidogenesis [2,3]. When partially-purified preparations of the enzyme are incubated in vitro with cyclic AMP, cyclic AMP-dependent protein kinase and ATP-Mg2+ the activity of the enzyme increases 3-fold. This activation is blocked by the presence of the protein inhibitor of the cyclic AMP-dependent protein kinase [4]. This evidence strongly suggests that the activity of cholesterol ester hydrolase is regulated by covalent phosphorylation. However, proof of this requires that the enzyme protein be identified and phosphate shown to be incorporated into the protein. In addition to cholesterol ester hydrolase, adrenal cortex also contains significant levels of a triacylglycerol hydrolase, the activity of which is stimulated by ACTH [S] and there is evidence that both activities are catalysed by the same polypeptide [6]. Evidence to the contrary, such as the selective inactivation of cholesterol ester hydrolase by chlorpyrifos oxone and the differing solubilities of the two enzymes in ammonium sulphate [6] may be at least partly explained by the finding of a second distinct triacylglycerol hydrolase in adrenal cortex [7]. Cholesterol ester hydrolase has been purified 57-fold from bovine adrenal cortex [4]. The major