Hydrolysis of triolein, cholesterol oleate, and 4-methylumbelliferyl stearate by acid and neutral ester hydrolases (lipases) from pigeon adipose tissues: effect of cAMP-dependent protein kinase

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Abstract
The properties of ester hydrolases (lipases) in a pH 5.2 precipitate fraction from pigeon adipose tissue were determined in assays which have used a variety of different substrate preparations. Hydrolase activity measured with an ethanolic triolein substrate dispersion was characterized as having a single pH optimum of 7.5. In contrast, assays performed with a glycerol-dispersed triolein preparation resulted in a distinct shoulder of hydrolase activity at acid pH values in addition to a pH optimum of 7.5; addition of lecithin to the glycerol-dispersed triolein substrate preparation decreased hydrolase activity at neutral and alkaline pH values but allowed a distinct acid pH optimum (at pH 5) to be observed. Assays with glycerol-dispersed preparations of cholesterol oleate and the fluorogenic substrate, 4-methylumbelliferyl stearate (MU-stearate) (both containing lecithin) also demonstrated hydrolase activity with both acid (pH 4.5) and neutral (pH 7.5-8) pH optima. Preincubation of the pigeon adipose tissue pH 5.2 precipitate fraction with Mg2+, ATP and cAMP resulted in a time-dependent increase in triglyceride (TG) hydrolase activity determined at pH 7 with an ethanolic triolein emulsion. This cAMP-dependent activation could be blocked by the addition of skeletal muscle protein kinase inhibitor; the addition of exogenous protein kinase (PrK) could reverse this inhibition. A Mg2+-dependent deactivation of PrK-activated TG hydrolase was observed; the rate of deactivation was enhanced by the addition of an exogenous phosphoprotein phosphatase. A cAMP-dependent PrK-catalyzed activation of hydrolase activity measured at pH 7 could also be determined with glycerol-dispersed substrate preparations of triolein, cholesterol oleate and MU-stearate. Acid hydrolase activity (measured at pH 4.5-5 with glycerol-dispersed substrates) was not increased by preincubation with ATP, cAMP and PrK. In experiments with glycerol-dispersed substrates, the kinetic mechanism associated with activation of pigeon adipose tissue hydrolase(s) was due to an increase in Vmax with little or no change in substrate affinity.

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