Characterization of a triglyceride-lipase and its natural substrate from crustacean hepatopancreas

PASQUEVICH M. Y.; DREON M. S.; LAVARÍAS S.; HERAS H

San Miguel de Tucumán

Congreso; XLV Reunión Anual - Sociedad Argentina de Investigación en Bioquímica y Biologla Molecular; 2009

SAIB

The aim of this work was to purify and characterize a citosolic triglyceride-lipase (lipase) activity and its natural substrates from the hepatopancreas of Macrobrachium borellii. Purification was followed by a zymographic assay and the esterase activity was confirmed by the hydrolysis of 14C-TG. The optimal temperature and pH values were 36°C and 8.0, respectively. The kinetic characterization using pNPP as substrate showed a Michaelis-Menten behavior with a Vmax=3.4x10-4 umol.min-1.mg-1 and a Km=1.63 mM. A MW of 72 KDa was estimated by PAGE. The TGs species from M. Borellii hepatopancreas were studied by silver nitrate chromatography allowing the separation of several groups of increasing Rƒ hereafter named group I-IV. Fatty acid (FA) composition of each TG group was analyzed by capillary GC. Group I was the major PUFA-containing one (48.5%) followed by group II (45.5%). Group III was dominated by monounsaturated FA (58.3%), while group IV had both monounsaturated and saturated FA (50.0% and 45.6%, respectively). Finally substrate specificity of lipase was determined by competence of Group I and different non-radiolabeled TGs on the rate of hydrolysis of labeled triolein. The enzyme showed higher specificity toward TG PUFA-containing species than toward saturated TGs. This is the first purification and characterization of a lipase and its natural substrates in crustaceans.