Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca

Abstract

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.

Figure 1

SDS-PAGE (15%) and Immunoblot analysis of CoSPL. (A) Analysis of purified of pure CoSPL by SDS-PAGE (15%). Lane 1, molecular mass markers (Pharmacia); Lane 2, CoSPL solution (20 μg) obtained after Mono S chromatography; lane 3, purified CoSPL (15 μg) obtained after Mono Q chromatography. The gel was stained with Coomassie blue. (B) Immunoblot analysis, pure CoOPL (15 μg) (lane 1), CoTPL (15 μg) (lane 2), CoCPL (15 μg) (lane 3), CoDrPL (30 μg ) and CoSPL (30 μg) (lane 5) using anti-CoOPL serum at 1:500 dilution.

Figure 2

Comparison of same kinetic properties of OPL and SPL. (A) Kinetic of hydrolysis of tributyrin emulsion by OPL (22 U) [10]. (B) Kinetic of hydrolysis of tributyrin emulsion by SPL (22 U). Lipolytic activity was followed at pH 8.5 and 37°C in the absence or in the presence of a molar excess of ostrich pancreatic colipase = 5 and 4 mM NaTDC.

Figure 3

the activation of SPL by colipases from other species and the capacity of CoSPL to activate various pancreatic lipases. (A) Effect of varying amounts of stingray, dromedary, turkey and chicken pancreatic colipases on the activity of pure SPL in the presence bile salts at pH 8.5 and 37°C. Enzymatic activity of lipase (2.1 nM) was assayed in the presence of 6 mM NaTDC with increasing amounts of colipase. (open circle): CoCPL; (open square): CoDrPL; (black square): CoSPL; (black circle): CoTPL (B) Effect of varying amounts of stingray pancreatic colipase on the activity of pure lipases from various species in the presence bile salts at pH 8.5 and 37°C. Enzymatic activity of lipase (2.1 nM) was assayed in the presence of 6 mM NaTDC with increasing amounts of colipase. (open circle): CoCPL; (open square): CoDrPL; (black square): CoSPL; (black circle): CoTPL

Figure 4

Chromatography of stingray colipase on FPLC Mono-S Sepharose and FPLC Mono-Q Sepharose. (A) Chromatography of stingray pancreatic colipase on FPLC Mono-S Sepharose. The column (2.6 cm × 20 cm) was equilibrated with 100 mM acetate buffer, pH 4.5, containing 0.05% Triton X-100 and 2 mM benzamidine (buffer A); a linear salt gradient (0.1 to 0.4 M NaCl) in buffer A was applied to the column; gradient chamber 200 ml; 3 ml fraction; flow rate, 30 ml/h. (B) Chromatography of stingray pancreatic colipase on FPLC Mono Q Sepharose step. The column (2.6 cm × 20 cm) was equilibrated with 10 mM tris-HCl buffer, pH 8, containing 10 mM NaCl and 2 mM benzamidine (buffer B); a linear gradient (0.1 to 0.3 M NaCl) in buffer B was applied to the column; 3 ml fraction; flow rate, 30 ml/h.