Cytosolic cholesterol ester hydrolase in adrenal cortex
Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent interfered in the operation of the ion-exchange columns, and pure enzyme was not obtained. Hydrophobic chromatography was studied but it was found to be unsuitable for the purification of CEH under the conditions investigated. An affinity chromatography technique was developed using columns of glass beads coated non-covalently with cholesterol oleate. Some positive results were obtained but the low capacity of the columns combined with the low concentration of the enzyme in the tissue cytosol prevented further study of the purified activity. A further purification procedure utilising a non-ionic detergent and gradient sievorptive chromatography resulted in a 150-fold purification of CEH from bovine adrenocortical cytosol with a recovery of about 25%. The specific activity of the partially purified enzyme (CEHQ2 preparation) was approximately 60 nmol oleic acid produced.min 3.mg protein Labelling studies using the CEHQ2 preparation, [γ-32P] ATP and [1,3-3H] DFP suggested that the enzyme activity was associated with a protein with Mr approximately 84 000. A phosphoprotein phosphatase with Mr 35 000 wTas purified from bovine adrenocortical cytosol to a state approaching homogeneity. The purified 32 phosphatase was active when measured towards P-phosphoprotexn and p-nitrophenyl phosphate. The role of this phosphoprotein phosphatase in the modulation of cytosolic CEH activity was investigated, but the enzyme did not deactivate partially-purified CEH from bovine adrenocortical cytosol. Cytosolic CEH in rat adrenal was found to exhibit a diurnal variation in activity. The enzyme activity was significantly higher during the dark phase. Serum corticosterone concentration reflected this variation in CEH activity. In vivo experiments including acute ACTH administration and dexamethasone suppression of pituitary ACTH secretion suggested that ACTH was involved in the production and maintenance of the diurnal rhythm of rat adrenal cytosolic CEH activity. The protein components of bovine adrenocortical and rat adrenal lipid droplets were investigated. The lipid droplets from both species contained a major protein subunit with Mr 40 000 and several other minor proteins. The protein profile was similar in both species. The Mr 40 000 apoprotein was able to be phosphorylated in rat lipid droplets but not in bovine lipid droplets. The delipidated apoprotein was phosphorylated in both cases. Bovine adrenocortical lipid droplets contained about 4% by weight protein, but CEH was found to be only a minor component of the protein fraction.