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.

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A phosphoprotein phosphatase has been partially purified from rat epididymal fat pads by a procedure utilizing ammonium sulfate and ethanol precipitations and chromatography on DEAE-Sephadex A-50. The phosphatase was eluted from Sephadex G-75 columns with an apparent molecular weight of 28 000. The phosphoprotein phosphatase catalyzed the reversible deactivation of protein kinase activated chicken adipose tissue hormone-sensitive triglyceride lipase. Phosphatase activity measured with activated triglyceride lipase as substrate was completely dependent upon the presence of metal ions (Mg2+, Ca2+, or Mn2+) and was inhibited by inorganic phosphate and adenine nucleotides. The fat pad phosphatase increased the rate of activation of glycogen synthase in rat adipose tissue infranatant fractions from fed and 24-h fasted rats but had little or no effect on synthase activity in infranatant fractions from rats fasted for 48 h. Fasting had no effect on rat fat pad phosphatase activity measured with triglyceride lipase as substrate, but phosphatase activity was decreased in preparations from diabetic rats.

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