J. Biol. Chem., Vol. 261, Issue 17, 7717-7722, 06, 1986
Protein kinase-mediated activation of temperature-labile and temperature-stable cholesteryl ester hydrolases in the rat testis
ML Bailey and WM Grogan
Both temperature-stable and temperature-labile testicular cholesteryl ester
hydrolases are shown to be regulated by an endogenous cAMP- dependent
protein kinase activity. The temperature-stable form (Mr = 28,000) was
activated 3-fold by the endogenous kinase. This activation was completely
blocked by protein kinase inhibitor. Following purification by high
performance gel permeation chromatography, the temperature-stable form
could also be activated 2-fold by bovine heart protein kinase, type I. The
partially purified endogenous protein kinase, type I, which was completely
separated from hydrolase activity by ion exchange chromatography, increased
hydrolase activity 2-fold in the presence of optimal concentrations of
cAMP, ATP, and Mg2+. Cholesteryl ester hydrolase activity could be
stabilized indefinitely at -10 degrees C with the addition of 0.1 mM
thioglycolate, but not by other thiol reagents. In contrast, the endogenous
protein kinase activity was lost from 104,000 X g supernatants after 14
days. However, the property of activation could be restored by addition of
bovine heart protein kinase. The temperature-labile hydrolase (Mr = 72,000)
could be totally inactivated by a Mg2+-dependent, fluoride-sensitive
cytosolic factor and reactivated by cAMP-dependent protein kinase. These
observations strongly suggest that the inactivating factor is a
phosphoprotein phosphatase.
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