Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

• Published in: Fish physiology and biochemistry 1991-12, Vol.9 (4), p.361-368
• Main Authors: Harmon, J S, Michelsen, K G, Sheridan, M A
• Format: Article
• Language: English
• Subjects: biochemical composition; Brackish; enzymes; Freshwater; Marine; Oncorhynchus mykiss
• ISSN: 0920-1742; 1573-5168
• DOI: 10.1007/BF02265156

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring(14)C-oleic acid release from(14)C-triolein.(14)C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°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 Vmax=0.016 nmol/h/mg protein and that Km=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg(2+). 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.