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

Abstract

The properties of ester hydrolases (lipases) in a pH 5.2 precipitate fraction from pigeon adipose tissue have been 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 found to be due to an increase in Vmax with little or no change in substrate affinity.