PURIFICATION AND CHARACTERIZATION OF HEPATIC TRIACYLGLYCEROL LIPASE ISOLATED FROM RAINBOW-TROUT, ONCORHYNCHUS-MYKISS

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring C-14-oleic acid release from C-14-triolein. C-14-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15-degrees-C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200-400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40-43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V(max) = 0.016 nmol/h/mg protein and that K(m) = 0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg2+. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.