title: Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex.
abstract: Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.
mesh_terms: D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D002787:Sterol Esterase / Q000302:isolation & purification*
chemical_list: D000242:Cyclic AMP; D011494:Protein Kinases; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase