Biochemical Characterization and Molecular Modeling of Pancreatic Lipase from a Cartilaginous Fish, the Common Stingray (Dasyatis pastinaca).

In order to identify fish enzymes displaying novel biochemical properties, we have chosen the common stingray (Dasyatis pastinaca), one of the most primitive living jawed aquatic vertebrates as a starting biological material to purify a lipase. A stingray pancreatic lipase (SPL) was purified from delipidated pancreatic powder. The SPL molecular weight was around 55 kDa which is slightly higher than that of known classical pancreatic lipases (50 kDa). This increase in the molecular weight was due to glycosylation. Like classic pancreatic lipases, SPL was found to be much more active on short-chain triacylglycerols than on long-chain ones. Natural detergents act as inhibitors of the SPL activity. This inhibition can be reversed by the addition of stingray colipase. Starting from total pancreatic messenger RNAs (mRNAs), partial stingray pancreatic lipase complementary DNA (cDNA) was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into the PGEM-T vector. Partial amino acid sequence of the SPL was homologous to that of Japanese eel, porcine, and human pancreatic lipases. A 3D structure model of the sequenced part of SPL was built using the 3D structure of porcine pancreatic lipase as template, since both lipases shared an amino acid sequence identity of 60%.

Lipolytic activity levels and colipase presence in digestive glands of some marine animals.

Studies on the digestive secretions in aquatic animals can elucidate certain aspects of their nutritive physiology. The aim of the present study was to compare the digestive lipase and phospholipase activities in ten marine species belonging to four classes following the taxonomic classification of marine organisms. All aquatic digestive tissues tested are equipped with lipase and phospholipase activities, assuming the hydrolysis of fat-rich food. The lipolytic activities determined in the pancreases of cartilaginous fishes were greater than those in bony fishes, molluscs and crustaceans. This finding might be explained by the strong digestive utilization of fat-rich macronutrients by these carnivorous fishes. A trend of activities and stabilities at different pH and temperatures for crude lipases and phospholipases from these aquatic animals suggests that the optimum pH and temperature for marine lipases are species dependent. Interestingly, the sardine caecal lipase and phospholipase were found to be mostly stable in a broad range of acidic pH values. The maximum activities of lipolytic enzymes from the hepatopancreases of Hexaplex trunculus (molluscs) and Carcinus mediterranus (crustaceans) were found to be 50 and 60 °C, respectively, whereas the optimal temperature of lipolytic enzymes for the other species was classically around 40 °C. Thermoactivity of molluscs' lipolytic preparations makes them potential candidates in industrial applications. Among digestive glands studied, only pancreas (cartilaginous fish) contained the classically known colipase. Regarded as the most primitive living jawed vertebrates, cartilaginous fishes represented by sharks and rays could be considered as the oldest vertebrates possessing a complex digestive system like that of mammals.

Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca.

BACKGROUND: Pancreatic colipase is a required co-factor for pancreatic lipase, being necessary for its activity during hydrolysis of dietary triglycerides in the presence of bile salts. In the intestine, colipase is cleaved from a precursor molecule, procolipase, through the action of trypsin. This cleavage yields a peptide called enterostatin knoswn, being produced in equimolar proportions to colipase. RESULTS: In this study, colipase from the common stingray Dasyatis pastinaca (CoSPL) was purified to homogeneity. The purified colipase is not glycosylated and has an apparent molecular mass of around 10 kDa. The NH2-terminal sequencing of purified CoSPL exhibits more than 55% identity with those of mammalian, bird or marine colipases. CoSPL was found to be less effective activator of bird and mammal pancreatic lipases than for the lipase from the same specie. The apparent dissociation constant (Kd) of the colipase/lipase complex and the apparent Vmax of the colipase-activated lipase values were deduced from the linear curves of the Scatchard plots. We concluded that Stingray Pancreatic Lipase (SPL) has higher ability to interact with colipase from the same species than with the mammal or bird ones. CONCLUSION: The fact that colipase is a universal lipase cofactor might thus be explained by a conservation of the colipase-lipase interaction site. The results obtained in the study may improve our knowledge of marine lipase/colipase.

Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca.

BACKGROUND: Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. RESULTS: A marine stingray phospholipase A2 (SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. CONCLUSIONS: SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.