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PubMed1979

Lipase and colipase in canine pancreatic juice as etiologic factors in fat necrosis.

Lipase and colipase have been purified to homogeneity from canine pancreatic juice. The purity of the lipase and colipase preparations was established by acrylamide gel electrophoresis. Either lipase or colipase alone did not produce fat necrosis when injected intraperitoneally into mice. Fat necrosis was seen only when both lipase and colipase were used together. Selective removal of lipase from fresh canine pancreatic juice by immunoprecipitation with an antilipase specific antiserum also eliminated its fat necrotizing activity. Together, these results identify the fat necrotizing factors to be pancreatic lipase and colipase. Their uncontrolled release during acute pancreatitis is believed to constitute the cause of fat necrosis. The absolute amount of lipolytic activity was not found to be the crucial factor in the induction of fat necrosis. It is suggested that the colipase molecule may have other functions besides enhancing the lipolytic activity of purified lipase in causing fat cell necrosis.

Animals↗
PubMed1978

Lipase-colipase interactions during gel filtration. High and low affinity binding situations.

The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.

Animals↗
PubMed1981

Importance of the N-terminal sequence in porcine pancreatic colipase.

Colipase exists in pancreatic juice in a pro-form which is activated by limited trypsin hydrolysis. During this activation, the N-terminal pentapeptide 1Val-Pro-Asp-Pro-5Arg is cleaved. The new N-terminal sequence formed, 6Gly-Ile-Ile-Ile-10Asn, contains three isoleucine residues. The importance of these for stimulating lipase activity has been investigated by successive Edman degradation of epsilon-acetimidolysine residues followed by limited trypsin hydrolysis. The epsilon-amidinated colipase obtained was fully active both with a phospholipid-covered triacylglycerol (Intralipid) and tributyrin as substrate. After removal of the three isoleucine residues, the activity of colipase was lost with Intralipid but not with tributyrin as substrate. The shortened colipases regained their Intralipid activity upon addition of long-chain fatty acids. The binding of colipase to lipase was not affected by removal of the isoleucine residues.

Amino Acid Sequence↗
PubMed1981

The identity and properties of two forms of activated colipase from porcine pancreas.

Colipase is excreted as a procolipase, colipase101. It is activated by low concentrations of trypsin, hydrolyzing the N-terminal pentapeptide. With higher concentrations of trypsin or in the presence of Ca2+ a smaller form of colipase containing 85 amino acids, appears. It has glycine as the N-terminal and arginine as the C-terminal amino acid residue and has lost 11 amino acids in the C-terminal chain. The ability of colipase85 to activate lipase with tributyrin as substrate is about the same as for colipase96 and procolipase. With fenfluramine, an anoretic agent, added to the tributyrin colipase assay system, the specific activities of colipase96 and colipase85 are similar and about five times higher than that of colipase101. With intralipid as substrate colipase85 enables lipase to reach the triacylglycerol substrate more rapidly than colipase96, having about six times shorter lagtime for a given concentration. Colipase84, obtained by splitting off the C-terminal arginine from colipase85, has a lagtime somewhere between colipase85 and colipase96, pointing to the importance of arginine85 for Intralipid activity. The binding between lipase and colipase has about the same strength for procolipase, colipase96 and for colipase85, Kd being about 10(-6) M either in buffer or in the presence of 2 mM taurodeoxycholate at pH 7. Addition of long chain fatty acids in the presence of bile salts increases the binding strength between colipase and lipase 100-fold, both for colipase96 and colipase85.

Animals↗
PubMed1984

Studies on chicken pancreatic lipase and colipase.

Lipase and colipase have been purified to homogeneity from chicken pancreatic tissue. The enzyme has a molecular weight (48 000) and catalytic properties similar to those of pancreatic lipase from higher mammals. Hydrolysis of triolein by chicken lipase is strongly inhibited by various bile salts, including sodium taurochenodeoxycholate, which is present in large proportion in chicken bile. Inhibition is reversed by colipase. With triolein as enzyme substrate, in the presence of sodium deoxycholate, no difference was observed in the ability of pure colipase from chicken, horse or pig to fully activate bile-salt-inhibited lipase from the same species. However, kinetic studies of the hydrolysis of a lecithin-stabilized emulsion of triacylglycerol (Intralipid) by chicken lipase show that the lag period is much longer in the presence of porcine colipase than with the chicken cofactor. This might reflect the higher ability of the avian enzyme to associate with colipase from the same species than with mammalian cofactors when the triacylglycerol substrate surface is covered with amphiphilic lecithin. From our study, the chicken pancreatic lipase/colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. It is then likely that they are of comparable physiological significance in fat digestion in avian and mammalian species.

Animals↗
PubMed1990

Effects of human pancreatic lipase-colipase and carboxyl ester lipase on eicosapentaenoic and arachidonic acid ester bonds of triacylglycerols rich in fish oil fatty acids.

Fish oil chylomicrons, obtained from mesenteric duct chyle of rats fed [3H]20:5 and [14C]20:4 or [3H]20:5 and [14C]18:2 in a fish oil emulsion, were incubated with human pancreatic lipase-colipase, human carboxyl ester lipase (CEL) and human duodenal contents. With duodenal contents, the triacylglycerols labelled with [3H]20:5 and [14C]20:4 were rapidly converted to free fatty acids (FFA) and monoacylglycerols. Also during incubation with lipase-colipase the [3H]- and [14C]triacylglycerols disappeared completely and at equal rates, but in this case much [3H]20:5 and [14C]20:4 accumulated in diacylglycerols. When CEL was also added, the rate of disappearance of [3H]- and [14C]triacylglycerols increased and the radioactivity of diacylglycerols decreased markedly. During incubation of chylomicrons labelled with [3H]20:5 and [14C]18:2 with lipase-colipase, the rates of hydrolysis of [3H]- and [14C]triacylglycerols were similar, but more [3H]20:5 than [14C]18:2 accumulated in diacylglycerols. The accumulation of [3H]diacylglycerol was reduced by adding CEL. Also when fatty acids were analyzed by gas chromatography, 20:5 was enriched in remaining triacylglycerol and in diacylglycerol after incubation with lipase-colipase alone. The data thus indicate that both lipase-colipase and CEL participate in the hydrolysis of 20:5 and 20:4 ester bonds of dietary triacylglycerol.

Animals↗
PubMed1983

The interaction between pancreatic lipase and colipase: a protein-protein interaction regulated by a lipid.

Pancreatic lipase readily adsorbs to a triglyceride droplet. In the intestine the triglyceride droplets are covered with bile salt and phospholipids which will prevent the adsorption of lipase. In this situation the activity of lipase is restored by colipase, another pancreatic protein. Lipase and colipase in solution form a 1:1 molar complex. I emphasize the fact that the binding and conformation of the two proteins in the complex is dependent on the type of lipids present and suggest that this lipid-determined structure of the complex is responsible for the actual function of lipase/colipase. It determines whether colipase assists lipase in binding to the bile salt-covered triglyceride droplet as is the case with tributyrin as substrate, and whether colipase in addition activates lipase as is the case with a mixed trioctanoin/lecithin monolayer substrate. In other words, lipase activity is regulated by the combined action of colipase and the lipid substrate.

Colipases↗
PubMed1993

Cloning and characterization of rabbit pancreatic colipase.

1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides. 2. For its action it requires a coenzyme, colipase. 3. The molecular mechanisms of the interaction of these two are not fully understood. 4. Further, molecular events that regulate and influence lipid absorption are ill defined. 5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase. 6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence. 7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide. 8. Northern blot analysis revealed a distinct band around 0.5 kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level. 9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53-73). 10. Rabbit enzyme also retained the N-terminal pentapeptide of its preform. 11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of colipase from porcine and human pancreatic juice by immunoaffinity chromatography.

Pure colipase was prepared by immunoaffinity chromatography from porcine and human pancreatic juice. A single form of the porcine colipase was obtained, having the structural and biological properties of previously characterized porcine procolipase A. Two forms of activated colipase (N-terminal Gly) were isolated from human pancreatic juice by the same procedure. The existence of two forms of activated colipase might arise from rapid activation of a precursor form of human colipase during collection of the pancreatic juice.

Animals↗
PubMed1989

Inhibitory properties and antigenic specificity of monoclonal antibodies to pancreatic colipase.

To understand the mechanism by which colipase acts as a protein cofactor for anchoring pancreatic lipase at triacylglycerol/water interface, we have used an immunochemical approach. Ten monoclonal antibodies (Mabs) against porcine pancreatic procolipase were produced. Purified immunoglobulins and Fab fragments were studied for their capacity to inhibit colipase-dependent lipase activity. These studies were carried out by using procolipase, the secretory form of the cofactor, and its trypsin-treated form obtained by removal of the amino terminal pentapeptide by trypsin. Reactivities of Mabs with both forms of the cofactor were also studied by immunoenzymatic methods. Mabs 6.1, 49.20. 75.8, 270.13 and 419.1 were found to inhibit lipolysis by preventing the binding of procolipase or trypsin-treated colipase to the lipid substrate. Mab 72.11 inhibited procolipase binding but had no effect on trypsin-treated colipase. Mab 72.11 reacted with procolipase in ELISA but showed no reactivity with trypsin-treated colipase. Finally, preincubation of Mab 72.11 with porcine procolipase prevented specific cleavage at the Arg5-Gly6 bond by trypsin. It could be concluded, that the five first residues of procolipase are structural elements of the antigenic determinant recognized by Mab 72.11. Results of ELISA additivity tests (cotitrations) further indicated that epitopes for Mabs 6.1, 72.11, 270.13 and 419.1 and for Mabs 49.20 and 75.8 are located in two distinct antigenic regions of the procolipase molecule. It appears then that the lipid binding domain of the pancreatic lipase protein cofactor comprises two regions. The first region corresponds to the amino terminal fragment of the protein. The second region is likely identical with the peptide segment at position 51-59 as previously hypothesized from NMR and spectrophotometric studies. Studies carried out on procolipase chemically modified at tyrosine residues provided evidence that epitopes for Mabs 49.20 and 75.8 are in or close to the region which contains tyrosines at positions 55 and 59, and that the two peptide regions essential for interfacial binding are spatially adjacent in the procolipase and the trypsin-treated form of the cofactor. General conclusions are in accordance with the location of antigenic regions of procolipase determined by predictive methods.

Animals↗
PubMed2000

Interactions of bile salt micelles and colipase studied through intermolecular nOes.

Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase-colipase-bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse colipase is similar to that already described for pig colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles.

Amino Acid Sequence↗
PubMed1990

Synthesis and characterization of the dansyltyrosine derivatives of porcine pancreatic colipase.

Steady-state and time-resolved fluorescence techniques were used to study dansyltyrosine derivatives of porcine pancreatic colipase. Nitration, reduction, acylation, and dansylation reactions were utilized to synthesize two fluorescently labeled colipases: (o-aminodansyltyrosine 55 porcine colipase) (DNStyr55PC) and o-aminodansyltyrosine 59 porcine colipase (DNStyr59PC). DNStyr55PC was 200% active, while the DNStyr59 derivative maintained 80% activity in a pH stat assay. Emission spectra, lifetime analysis, acrylamide quenching, polarization, and anisotropy decay studies indicated that Tyr55 was located on the solvent-exposed surface of the protein, where the fluorophore experienced free rotation. Identical experiments done on DNStyr59PC indicated that Tyr59 was in a partially buried environment and the motion of the dansyl tyrosine group was hindered. The double-exponential decay of the fluorescence emission of N-acetyl-o-aminodansyltyrosine ethyl ester (DNStyr) and the DNStyr derivatives of colipase was investigated with pH, temperature, solvent, and emission-resolved-lifetime experiments. The existence of excited-state processes was eliminated in both pH and emission-resolved-lifetime experiments, whereas temperature studies indicated either a rotational isomer or a differential solvent quenching mechanism for multiple decay kinetics. These experiments also showed that DNStyr was a sensitive probe of solvent polarity and viscosity, but not of pH.

Animals↗
PubMed1975

Interactions of colipase with bile salt micelles. 2. Study by dialysis and spectrophotometry.

The finding reported in the preceding paper that colipase is able to bind one sodium taurodeoxycholate micelle per molecule was confirmed by dialysis and spectrophotometry. Dialysis in the presence of labelled sodium taurodeoxycholate provided a direct qualitative proof of taurodeoxycholate binding to colipase. This binding was found to occur only above the critical micelle concentration. But, dialysis did not give any information about the composition of the associations, because equilibrium was not attained at the end of the assays. Addition of sodium taurodeoxycholate above the critical micelle concentration was also observed to induce a strong perturbation of the ultraviolet spectrum of one or several of the three tyrosines of colipase. The variation of the perturbation as a function of sodium taurodeoxycholate concentration was consistent with the binding of a single micelle to colipase. The dissociation constant calculated in "micelle molarity" was approximately 1 X 10(-4) M. The colipase-bile salt micelle association can fix one molecule of lipase to form a ternary complex which represents an interesting model of a protein-protein interaction mediated by an organized lipid structure. The ternary complex is probably also a model for lipase-substrate interactions in the presence of an amphipath.

Binding Sites↗
PubMed2006

Biochemical and structural comparative study between bird and mammal pancreatic colipases.

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.

Amino Acid Sequence↗
PubMed1977

Effects of colipase on hydrolysis of monomolecular films by lipase.

In a system free of bile salts we measured lipase hydrolysis of 1,3-didecanoylglycerol films in the presence or absence of colipase at different surface pressures. The strong, but not absolutely specific protective effect of colipase, most visible at low surface pressure, can account for the higher enzyme activity in the presence of colipase. This can be understood by taking into account simultaneous penetration and surface inactivation fluxes. Using radioactively labeled lipase, we have shown for the first time in a bile salt-free system that the critical surface pressure above which lipase can no longer penetrate a 1,2-didodecanoylphosphatidylglycerol monlayer is around 23 dynes/cm. Colipase increased this critical surface pressure to 30 dynes/cm indicating that it enables lipase penetration between 23 and 30 dynes/cm. The transfer experiment showed that colipase acts by first penetrating the lipid film and then serving as an anchor for lipase into the film.

Colipases↗
PubMed1998

The hydrophobic surface of colipase influences lipase activity at an oil-water interface.

The interaction of pancreatic triglyceride lipase and colipase at an oil-water interface is required for efficient digestion of dietary fats and provides a model system for the interaction of proteins at biological membranes. Colipase has two important surfaces, a hydrophilic surface that interacts with lipase and a hydrophobic surface that presumably interacts with substrate. To begin our investigations into the role of the hydrophobic surface in the function of colipase, we replaced three neighboring tyrosine residues at positions 55, 58, and 59 in the hydrophobic surface with aspartic acid. Two of the three residues, Tyr55 and Tyr59, influenced the activity of colipase. Introducing aspartic acid at either position decreased the activity with long-chain triglycerides, but not with a short-chain triglyceride. Decreased ability of the mutants to anchor lipase to long-chain triglycerides did not explain the altered activity of the mutants. A mutant containing aspartic acid at positions 55 and 59 had no activity with any substrate and did not anchor lipase to either short- or long-chain triglycerides. These results identify the two tyrosine residues that interact with substrate and suggest that the hydrophobicity of the surface containing these tyrosines influences colipase function and the substrate selectivity of pancreatic triglyceride lipase.

Aspartic Acid↗
PubMed1987

Zonal high-performance affinity chromatography as a tool for protein interaction studies with special reference to the lipase-colipase complex.

The technique of zonal high-performance affinity chromatography applied to the lipase-colipase system (lipase B as eluted acceptor and colipase as silica-bonded ligand) gave qualitatively the same results as conventional affinity chromatography. The elution volume of the acceptor increases with decreasing load introduced at constant volume into the column of ligand-bonded silica. This led to the use of a mathematical treatment for calculating the dissociation constant (KD) of the lipase-colipase complex. The influence of some physical and chemical chromatographic parameters was studied. Increasing temperature and flow-rate reduced the affinity of lipase for colipase, whereas it was only slightly modified by increasing the ionic strength. The KD value was minimal and equal to 0.1 X 10(-6) M at pH 4.7 and 0.38 X 10(-6) M at pH 6.5, after correction for the flow-rate. The latter value is similar to that obtained by more conventional techniques. The absence of some marked KD modifications by ionic strength and the value of delta S for the complex association obtained by temperature studies suggest the intervention of mixed hydrophobic-ionic interactions in the formation of the lipase-colipase complex. Their respective importances are discussed.

Animals↗
PubMed2001

Do human bile salt stimulated lipase and colipase-dependent pancreatic lipase share a common heparin-containing receptor?

Bile salt stimulated lipase (BSSL), a lipolytic enzyme secreted with pancreatic juice and with human milk, is in concert with colipase-dependent pancreatic lipase, important for the intestinal digestion of dietary lipids. BSSL may also facilitate uptake of free cholesterol from the intestinal lumen, while colipase-dependent lipase has a similar role for fatty acids. According to this theory, the two lipases bind to the intestinal mucosa via a common heparin-involving receptor. In the present study, binding of the two lipases to heparin was explored in vitro using purified human lipases and heparin molecules varying in both chain length and charge density. Native, but not denatured, BSSL bound avidly to heparin and several of the heparin variants. In contrast, at physiologic salt concentration, colipase-dependent lipase did not bind to heparin. Thus, our data do not support the view that the two lipases share a common intestinal heparin-like receptor. Hence, it seems unlikely that such binding could be of physiologic relevance for colipase-dependent lipase, although for BSSL the data are supportive.

Albumins↗