Protein kinase-mediated activation of temperature-labile and temperature-stable cholesteryl ester hydrolases in the rat testis.
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.
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000201:enzymology; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D002787:Sterol Esterase / Q000378:metabolism*; D013737:Testis / Q000201:enzymology*; D013816:Thermodynamics
D004492:Edetic Acid; D011494:Protein Kinases; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase