Journal of Lipid Research, Vol 19, 561-569, Copyright © 1978 by Lipid Research, Inc.
Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex
S Naghshineh, CR Treadwell, LL Gallo and GV Vahouny
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.
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