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PubMed1980

Interaction of porcine pancreatic colipase with a nonionic detergent, Triton X-100: spectrophotometric studies.

Strong perturbation of the ultraviolet spectrum of the tyrosines of porcine pancreatic colipase A is observed in the presence of Triton X-100 at concentration above the critical micellar concentration. Spectrophotometric titration of the phenolic groups of the protein shows that the apparent pKa value for two tyrosines is about 10.3, while the third tyrosine has a higher pKa value above 11.6. This residue is still protonated at pH 13 in the presence of Triton X-100. All perturbations induced by the nonionic detergent can be interpreted as resulting from interactions between colipase and Triton X-100 molecules at a hydrophobic site of the protein that includes the tyrosine residues. Results obtained in studies with Triton X-100 are similar to those already reported by Sari et al. (Eur. J. Biochem. 58:561 (1975) on the interaction of colipase with taurodeoxycholate. It is likely that the binding of both types of detergent occurs at the same specific site on the protein molecule. Data presented in this communication give further support to the hypothesis that a hydrophobic domain (residues 49-57), including all three tyrosines of the colipase molecule, participate to the well characterized interaction of the lipase cofactor with triglycerides at lipid-water interfaces.

Animals↗

PubMed1982

The temperature-dependent interfacial inactivation of porcine pancreatic lipase. Effect of colipase and bile salts.

This paper confirms and extends the previous observation that colipase and bile salts stabilize pancreatic lipase against inactivation at its water/substrate interface. It is shown that colipase and bile salts above their critical micellar concentration offer better protection than either of them alone. Colipase has no effect on the catalytic efficiency of lipase against an emulsified substrate in the absence or presence of bile salts. Its reported activation of pancreatic lipolysis at high temperatures in the absence of bile salts is, most likely, fully explained by its protective effect on lipase inactivation. Colipase at high concentrations relative to lipase inhibits the enzyme activity in a competitive fashion. The temperature-dependent surface inactivation of lipase has certain consequences for the methodology of lipase activity determination.

Animals↗

PubMed1986

Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase.

Mixed micelles of bile salt and phospholipids inhibit the lipase-colipase-catalysed hydrolysis of triacylglycerols. Free fatty acids can reverse this inhibition and reactivate lipase-colipase. This reactivation is either due to the formation of a high-affinity complex between lipase and colipase induced by free fatty acids and/or to a change of the quality of the interface. Lauric acid, oleic acid and linoleic acid are the most potent reactivators, while short-chain free fatty acids have no effect and long-chain, saturated free fatty acids inhibit the lipase-colipase activity further. The physiological relevance of these results is evident as the glyceride emulsion reaching the duodenum already contains free fatty acids due to the activity of lingual lipase in the stomach.

Animals↗

PubMed1994

Molecular cloning and expression of two horse pancreatic cDNA encoding colipase A and B.

Pancreatic colipase plays an essential role in the intestinal fat digestion by anchoring lipase on lipid/water interfaces in the presence of bile salts. In contrast to other species, two molecular forms of colipase, A and B, have been found in horse. The two corresponding cDNAs were isolated from a horse pancreatic library and their nucleotide sequences were determined. Moreover, for the first time, active colipase has been obtained after transfection of COS cells by either colipase A or B cDNA.

Amino Acid Sequence↗

PubMed1994

Digestion of triacylglycerols containing long-chain polyenoic fatty acids in vitro by colipase-dependent pancreatic lipase and human milk bile salt-stimulated lipase.

To assess the role of human milk bile salt-stimulated lipase (BSSL) in the digestion of polyunsaturated ester bonds of triacylglycerols, hydrolysis of docosahexaenoic acid (22:6(n-3)) ester bonds was compared to that of oleic acid (18:1(n-9)) or arachidonic acid (20:4(n-6)) esters. As model substrates, we used rat chylomicrons obtained after feeding human milk fat globules and radiolabeled fatty acids. Radiolabeled chylomicrons were incubated with colipase-dependent pancreatic lipase, with BSSL, or with both enzymes in combination. Both enzymes hydrolyzed 18:1 more efficiently than 22:6 esters. With colipase-dependent lipase there was a large accumulation of 22:6 in diacylglycerol whereas with BSSL it accumulated mainly in monoacylglycerol. Esters containing 20:4 were hydrolyzed by BSSL as efficiently as 18:1 but this fatty acid also accumulated as diacylglycerol with colipase-dependent lipase. At low bile salt concentrations, as found in duodenal contents of newborns, colipase-dependent lipase was virtually unable to hydrolyze esters of 20:4 and 22:6 whereas BSSL hydrolyzed these esters at appreciable rates. Combining the two enzymes gave the most efficient hydrolysis of all fatty acids tested regardless of bile salt concentrations. BSSL may thus have a physiological role in completing duodenal hydrolysis of milk triacylglycerols containing 22:6- or 20:4-esters to free fatty acids and monoacylglycerol.

Arachidonic Acid↗

PubMed1981

Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies.

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of bile salt-inhibited lipase with emulsified triolein in the absence and in the presence of lecithin show that tryptic hydrolysis of the protein cofactor increases its affinity for the enzyme in the presence of lipid substrate. In both cases, it was found that the apparent dissociation constant of the lipase-colipase complex is decreased by one order of magnitude. Our results confirm that the biological activity of the lipase cofactor is enhanced by specific tryptic cleavage in the amino terminal region of the polypeptide and support the suggestion by Borgström et al. (Borgström, B., Wieloch, T., Erlanson-Albertsson (1981) FEBS. Lett. 108, 407-410) that the secreted form of colipase is a precursor.

Animals↗

PubMed1990

Rat pancreatic colipase mRNA: nucleotide sequence of a cDNA clone and nutritional regulation by a lipidic diet.

A cDNA clone encoding rat pancreatic colipase was isolated using as a probe a synthetic deoxyoligonucleotide corresponding to a highly conserved amino acid sequence region in colipases from other species. The cloned messenger codes for a protein of 95 amino acids plus a signal peptide of 17 amino acids. The structure of the full-length cDNA was also determined and the corresponding amino acid sequence showed a high degree of homology with those of other known colipases. Quantification of the homologous mRNA in the pancreas of animals fed a high-lipid diet was consistent with a specific though moderate induction of colipase messenger by the nutritional manipulation.

Amino Acid Sequence↗

PubMed1992

Immunochemical studies of pancreatic colipase-lipase interaction employing immobilized synthetic peptides.

In view to study the possible participation of the sequence portions of colipase including or close to the free carboxyl groups at positions 15 and/or 72 to the binding with pancreatic lipase, we have used three synthetic peptides matching portions 8-16, 59-67 and 67-72 of the amino acid sequence. Polyclonal rabbit anticolipase immune serum, which cross-reacts with peptides in ELISA, was fractionated on columns of peptide coupled to Sepharose. Of the three fractions of antibodies, only that interacting with peptide 8-16 had the capacity to inhibit colipase-dependent lipase activity by specifically preventing the association of lipase with its protein cofactor previously bound to lipid. We conclude that the region spanning residues 8-16 of colipase is of importance for colipase-lipase interaction in the active complex formed at interface.

Amino Acid Sequence↗

PubMed1984

The primary sequence of human pancreatic colipase.

The amino acid sequence of an activated colipase purified from human pancreas was determined. The protein consists of a single polypeptide chain of 86 amino acids (human colipase86) and has a molecular weight of 9289. The sequence was determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of two CNBr peptides. Sequence determination of porcine procolipase II was also performed, which showed that in the original sequence determination apparently two residues were missed. These residues were determined to be a leucine at position 37 and a serine in position 50. For comparison with porcine and equine procolipases, the residues composing human colipase are numbered from 6 to 91. No human procolipase has been isolated so far. The colipases from man, pig, horse and chicken show a high degree of homology: human colipase differs from the other proteins by substitutions of 19 (porcine), 24 (equine A) and 21 (equine B) residues, respectively.

Amino Acid Sequence↗

PubMedDate not supplied

Further results on lipase-colipase interactions studied by affinity chromatography.

Affinity chromatography of lipase on a colipase-coupled gel was studied in the present paper. The elution volume of the associable lipase increased when the loaded amount decreased. A KD value of 1.9 X 10(-6) M at pH 6.2 was thus deduced. A minimum value of 1.5 X 10(-6) M was obtained at pH 5.1-5.3. Mixed micelles associated with coupled colipase, but no modifications of lipase-colipase interactions took place when mixed micelles were added to the elution buffer. DMMA-modified coupled colipase failed to interact with lipase, owing to the specific orientation of the modified cofactor in the gel.

Animals↗

PubMed1988

Minireview on pancreatic lipase and colipase.

By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.

Amino Acid Sequence↗

PubMed1988

A possible physiological function of pancreatic pro-colipase activation peptide in appetite regulation.

Pancreatic pro-colipase activation peptide, a pentapeptide with the sequence VPDPR was found to significantly suppress food intake of 20 h fasted Sprague-Dawley rats in a dose-dependent way. A rat treated with pro-colipase-enriched pellets for 26 days showed decreased daily food intake and retarded growth, which were restored during a following period of regular feeding. Genetically obese Zucker rats (fa/fa) were found to contain a reduced content of pancreatic pro-colipase (60% reduction), whereas the pancreatic lipase content was normal. A physiological function of pancreatic pro-colipase activation peptide as an endogenous satiety signal is suggested.

Animals↗

PubMed1992

Separation and characterization of the precursor and activated forms of porcine and human pancreatic colipase by reversed-phase liquid chromatography.

Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.

Animals↗

PubMed1975

On the formation of a ternary complex between lipase, colipase and micelles of amphipathic compounds.

Pancreatic lipase, colipase and 1-glyceryl octyl ether or lysolecithin micelles were shown to form a ternary complex probably resulting from the fixation of the enzyme to the binary colipase-micelle association already well documented in the case of bile salt micelles. Kinetic assays show that the colipase favours the lipase activity on tributyrin emulsions in presence of 1-glyceryl octyl ether. The UV spectrum of a derivative of lipase in which two 2-hydroxy 5-nitrobenzyl groups are attached to tryptophan, is distinctly modified by addition of 1-glyceryl octyl ether micelles in the presence of colipase.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed2002

Activation of horse PLRP2 by bile salts does not require colipase.

Although structurally similar to pancreatic lipase (PL), the key enzyme of intestinal fat digestion, pancreatic lipase-related protein type 2 (PLRP2) differs from PL in certain functional properties. Notably, PLRP2 has a broader substrate specificity than PL, and unlike that of PL, its activity is not restored by colipase in the presence of bile salts. In the studies presented here, the activation mechanism of horse PLRP2 was studied through active site-directed inhibition experiments, and the results demonstrate fundamental differences with that of PL. The opening of the horse PLRP2 flap occurs as soon as bile salt monomers are present, is accelerated in the presence of micelles, and does not require the presence of colipase. Moreover, in contrast to PL, horse PLRP2 is able to directly interact with a bile salt micelle to form an active binary complex, without the micelle being presented by colipase, as evidenced by molecular sieving experiments. These findings, together with the sensitivity of the horse PLRP2 flap to partial proteolysis, are indicative of a higher flexibility of the flap of horse PLRP2 relative to PL. From these results, it can be concluded that PLRP2 can adopt an active conformation in the intestine, which could be important for the further understanding of the physiological role of PLRP2. Finally, this work emphasizes the essential role of colipase in lipase catalysis at the lipid-water interface in the presence of bile.

Animals↗

PubMed1997

Effects of colipase and bile salts on the catalytic activity of human pancreatic lipase. A study using the oil drop tensiometer.

Using the oil drop technique, we studied the effects of colipase and bile salts on the rate of hydrolysis of soybean oil by human pancreatic lipase (HPL) as well as on the interfacial binding. Upon continuously recording the decrease in the interfacial tension with time, a 10-15-fold increase in the HPL activity was found to occur in the presence of colipase. The catalytic rate constants of hydrolysis measured at the oil drop surface were found to be of the same order of magnitude as those obtained with monomolecular films spread at the air-water interface. Biotin-labeled HPL (HPL*) was used to determine the amount of adsorbed enzyme using an ELISA test. Less than 1% of the total amount of injected HPL* molecules was found to have adsorbed to the oil-water interface, and no significant effects of colipase on HPL* binding were observed. No significant changes in the hydrolysis rates or the binding of HPL* were detected in the presence of bile salts at concentrations ranging from below their critical micellar concentration (CMC) up to 100 microM. At the oil-water interface, in the absence or presence of bile salts below their CMC, it can be concluded that the colipase is a true lipase cofactor, i.e, it increases the enzyme turnover (approximately 10-15-fold) and does not affect the interfacial lipase adsorption.

Animals↗

PubMed1996

Lipase activation by nonionic detergents. The crystal structure of the porcine lipase-colipase-tetraethylene glycol monooctyl ether complex.

The crystal structure of the ternary porcine lipase-colipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 A resolution. The crystals belong to the cubic space group F23 with a = 289.1 A and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C.(1993) Nature 362, 814-820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4 degrees rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.

Animals↗

PubMed2006

Val-407 and Ile-408 in the beta5'-loop of pancreatic lipase mediate lipase-colipase interactions in the presence of bile salt micelles.

In a previous study, we demonstrated that the beta5'-loop in the C-terminal domain of human pancreatic triglyceride lipase (hPTL) makes a major contribution in the function of hPTL (Chahinian et al. (2002) Biochemistry 41, 13725-13735). In the present study, we characterized the contribution of three residues in the beta5'-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the beta5'-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the beta5'-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the beta5'-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the beta5'-loop.

Adsorption↗