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PubMed1975

Interactions of colipase with bile salt micelles. 1. Ultracentrifugation studies.

A detailed investigation by ultracentrifugation of the colipase-taurodeoxycholate system showed the formation of well-defined mixed associations with a sedimentation coefficient of about 2.2S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor. Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.

Binding Sites↗
PubMed1981

Inhibition of human pancreatic lipase-colipase activity by mixed bile salt-phospholipid micelles.

Mixed dihydroxy bile salt-phosphatidylcholine (PC) micelles can inhibit the hydrolysis of gum arabic-stabilized long-chain triglyceride emulsions by 10(-8) to 10(-9) M concentrations of human pancreatic lipase and colipase. Trypsin treatment of this colipase preparation did not reverse the inhibition, suggesting that procolipase, as a possible contaminant, was not the inhibitory factor. Human biliary phospholipid-cholesterol liposomes, isolated by gel filtration and redissolved in bile salt solutions, inhibited lipolysis to the same degree as solutions of bile salt containing purified PC. The degree of inhibition depended principally on the species of bile salt present (e.g., taurochenodeoxycholate greater than taurodeoxycholate greater than tauroursodeoxycholate greater than taurocholate). In the absence of bile salt, PC (0.4 mM) liposomes alone were not inhibitory over the physiological time range studied. Bile salt solutions of phosphatidylethanolamine or sphingomyelin also inhibited lipase activity, whereas those containing oleyl alcohol, oleyl aldehyde, oleic acid, and lyso-PC did not. PC molecules were found to partition between the triglyceride emulsion interface and the bulk aqueous phase. Full reversal of inhibition occurred in the presence of phospholipase A2, which hydrolyzed the phospholipids to lysolecithin and fatty acids. Mixed bile salt-phospholipid micelles caused marked decrease in the binding of lipase and colipase to the triglyceride substrate and displaced the proteins into the aqueous phase. The results taken together suggest that colipase binds to certain bile salt-PC associations independent of whether the aggregates are located at the surface of a triglyceride particle as a monolayer or in the bulk aqueous phase as mixed micelles.

Bile Acids and Salts↗
PubMed1981

Hydrolysis of human milk fat globules by pancreatic lipase: role of colipase, phospholipase A2, and bile salts.

Human milk fat globules were used to explore how dietary triglycerides are hydrolyzed by pancreatic lipase. These triglycerides were hydrolyzed very slowly by lipase alone as if the surface layer of proteins and phospholipids impeded the action of the enzyme. The inhibition of lipase activity could be overcome by addition either of colipase or of pancreatic phospholipase A2. Colipase enhanced triglyceride hydrolysis in a dose-dependent manner whether bile salts were present or not. Bile salts had no effect on the activity of pancreatic lipase alone but further enhanced the activity at all concentrations of colipase tested. Bile salts were a prerequisite to relieve inhibition of lipase activity by phospholipase A2. Human milk fat globules exposed to phospholipase A2 should be representative of a physiological substrate for pancreatic lipase. A major new observation was that bile salts, even at high concentrations, stimulated triglyceride hydrolysis of such phospholipase-treated globules by pancreatic lipase also in the absence of colipase.

Bile Acids and Salts↗
PubMed1976

Effects of colipase and taurodeoxycholate on the catalytic and physical properties of pancreatic lipase B at an oil water interface.

A monolayer reaction system employing tripropionin and siliconized glass beads was used to study the effects of taurodeoxycholate and colipase on the catalytic activity, interfacial stability, and interfacial affinity of porcine pancreatic lipase B (EC 3.1.1.3) The stability and catalytic activity of lipase at the bead-water interface are governed by the same two ionizable groups with pKa values (in the absence of cofactors) of 5.6 and 9.3. Colipase alone or with bile salt caused only a slight perturbation of these values. At low concentrations, 0 to 0.3mM, taurodeoxycholate increases the stability of lipase by 5-fold. At higher concentrations, 0.3 to 0.8 mM, but still below its critical micelle concentration, taurodeoxycholate prevents the adsorption of lipase to the bead-water interface. This appears to be the major mechanism by which this bile salt inhibits lipolysis. Colipase exerts small positive effects on lipase stability and catalytic activity. More importantly, colipase enables the adsorption of lipase in the presence of bile salt, thereby reversing the inhibition.

Animals↗
PubMed1986

Processing of pro-colipase and trypsinogen by pancreatic dipeptidyl peptidase IV.

Purified dipeptidyl peptidase IV from porcine pancreas or from human placenta cleaves N-terminal dipeptides from two proteins of the pancreatic juice, namely trypsinogen and pro-colipase. Phenylalanyl-proline is very effectively released (Km approximately 50 microM) from bovine or porcine trypsinogen. Both purified dipeptidyl peptidases also rapidly cleave valyl-proline from the N-terminus of porcine pro-colipase. This degradation does not increase the colipase activity of the precursor. However, under certain conditions, which are not fully understandable at present, dipeptidyl peptidase IV releases more slowly a second dipeptide, aspartyl-proline, from pro-colipase, and this results in a partial activation. Dipeptidyl peptidase IV apparently lines the excretory ducts of porcine pancreas and, therefore, is in close contact to the proteins of pancreatic juice in vivo. The possible significance of these degradations is discussed.

Amino Acids↗
PubMed1975

On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts.

The interactions between pancreatic lipase and colipase and the substrate and the effect of bile salts on these interactions have been investigated by the use of kinetic experiments and studies on the semiquantitative phase distribution of lipase and colipase activities. The results suggest that lipase binds to hydrophobic interfaces with partial irreversible inactivation. Bile salts in the range of micellar concentrations and above a pH of about 6.5 displace lipase from this binding, resulting in a reversible in activation. At pH values below about 6.5, lipase binds strongly to the substrate even in the presence of bile salt, and a low activity peak is seen around pH 5.5. This is the result of the binding of lipase to the "supersubstrate" and the activity of the catalytic site. In the presence of bile salt, colipase promotes the binding of lipase to the "supersubstrate" but not to other hydrophobic interfaces, and catalytic activity is reestablished. Kinetic data indicate that the binding between colipase and lipase in the presence of substrate is strong and occurs in an approximately stoichiometric relationship.

Animals↗
PubMed1982

Development of pancreatic enzymes in fetal and suckling rats with emphasis on lipase and colipase.

Pancreatic amylase, chymotrypsinogen, lipase and colipase were assayed, at intervals, in rats from day 16 of fetal life until weaning. In the fetus, amylase and chymotrypsinogen accumulated regularly, in parallel, until birth. Lipase and colipase accumulation slowed down between day 20 and birth. The ratio of colipase to lipase was extremely high (9.5) and decreased until weaning towards adult values. Enzyme contents of the pancreas were depleted after birth and remained low until day 14. Intestinal concentrations were equally low, showing that pancreatic depletion was not due to hypersecretion. Protein synthesis was very active, intermediate between that of the fetus and of the adult. It is concluded that in the early suckling phase the proteins synthesized are mainly constitutive and not enzymatic. Starvation followed by refeeding showed that secretion sensitivity to nutritional stimulation only appears at 14 days. During the suckling period amylase concentrations decreased, evidencing a degree of nutritional sensitivity to the low level of carbohydrate in the diet. The productive capacity for lipase underwent a slow maturation which was not even complete at weaning, since concentrations had not yet reached adult level despite the high fat content of milk. This was in part compensated for by the high proportion of colipase but shows that lipase was not adaptative during this phase and that pancreatic lipase can hardly account for lipid digestion before weaning.

Amylases↗
PubMed1995

C-terminal domain of human pancreatic lipase is required for stability and maximal activity but not colipase reactivation.

Fungal lipases and human pancreatic lipase (hPL) share a common tertiary structure termed the alpha/beta hydrolase fold. In contrast, the region C-terminal to the common tertiary structure does not share any common structural features with fungal lipases, leading to the hypothesis that the divergent C-terminal domain confers specific properties to hPL. To study the role of the C-terminal domain in hPL function, we made substitution and deletion mutations in the C-terminal domain. The mutant proteins were expressed in transfected COS-1 cells and the secreted proteins were analyzed by immunoblot and for lipase activity. Substitution mutants in multiple lysine residues, in aspartate 390, or in tyrosine 404 did not affect secretion or lipase activity of the mutants. Significantly, the mutants still required colipase for maximal activity. Deletion of the C-terminal domain decreased the amount of truncated, mutant protein in the medium of transfected cells and decreased the specific activity of the mutants. Still, maximal activity required colipase, indicating that the deletion mutants interacted with colipase. Interfacial binding of the truncated deletion mutants was decreased relative to wild-type hPL. The newly synthesized deletion mutants were not as efficiently secreted from the transfected cells as wild-type hPL, and the mutant proteins that appeared in the medium were less stable than the wild-type hPL. These findings suggest that the C-terminal domain is required for proper folding or processing of hPL, confers stability, and increases activity, but is not absolutely required for colipase reactivation of the bile salt-inhibited enzyme.

Amino Acid Sequence↗
PubMed1982

Measurement of the binding of human colipase to human lipase and lipase substrates.

Equilibrium partition in an aqueous two-phase system was the method used for quantitative determinations of the binding between human colipase and human lipase and three triacylglycerol substrates: Intralipid tributyrin and triolein. The measurements were performed in a dextran/polyethyleneglycol system at pH 7.0 in the presence of 2 mM sodium taurodeoxycholate and 150 mM NaCL. The binding of colipase to lipase had a dissociation constant Kd = 4.8 . 10(-8) M. The dissociation constants for the binding of colipase to Intralipid, tributyrin and triolein were found to be 2.10(-7) M, 4.8 . 10(-8) M and 6.2 . 10(-8) M, respectively.

Colipases↗
PubMed1992

Direct involvement of the C-terminal extremity of pancreatic lipase (403-449) in colipase binding.

After a selective cleavage of a lipase/colipase cross-linked complex, the colipase has been shown to be bound to a 5 kDa lipase fragment identified as the C-terminal extremity of the chain extending from residue 403 to the C-terminus (Cys 449). The colipase binding site on lipase is therefore localized in a restricted contact area. Moreover, from sequence comparison of lipase from various species, an acidic residue, Glu 440, is likely to be involved in ion pairing with colipase.

Amino Acid Sequence↗
PubMed1994

Pancreatic triglyceride lipase and colipase: insights into dietary fat digestion.

Dietary fats have an impact on health and disease. A pancreatic exocrine protein, pancreatic triglyceride lipase, is essential for the efficient digestion of dietary fats. This enzyme requires another pancreatic exocrine protein, colipase, for full activity in the gut lumen. In addition to its importance in fat digestion, pancreatic triglyceride lipase has potential applications in medical therapy, medical diagnostics, and industry. This potential stimulated interest in lipases; radiograph during the last few years, studies applying the technologies of molecular biology and radiograph crystallography greatly increased our knowledge about pancreatic triglyceride lipase and colipase protein structure, enzyme mechanism, and gene structure. This review focuses on these recent advances and discusses models for the kinetic properties of pancreatic triglyceride lipase and for the interaction of pancreatic triglyceride lipase with colipase.

Amino Acid Sequence↗
PubMed1984

Purification and characterization of pancreatic colipase from the dogfish (Squalus acanthius).

Pure colipase from dogfish (Squalus acanthius) was obtained from an extract of pancreatic gland. It has a high isoelectric point (10.2) and the molecular weight was calculated to be 9108-9383. The N-terminal sequence was shown to be Gly-Leu-Phe-Leu-Asn-Leu-Ser-Ala-Gly-Glu-Leu-Cys-Val-Gly-Ser-Phe-Gln -Cys-Lys-Ser-Ser-Cys-Cys-Gln-Arg-Glu-Thr-Gly-Leu-Ser-Leu-Ala -Arg-Cys-Ala-. This sequence shows great homology with colipases from man, horse, pig and hen. There were indications of the existence of a proform of dogfish colipase. The propeptide was found to be Ala-Pro-Glu-Arg.

Amino Acid Sequence↗
PubMed1990

The interaction of bile salt micelles with the dansyltyrosine derivatives of porcine colipase.

The interaction of bile salt micelles with the tyrosines of pancreatic colipase was assessed by steady-state and time-resolved fluorescence techniques. Dansyltyrosine fluorescence showed that Tyr-55 was located in the proposed interface recognition site. In support of this claim was a 70 nm blue shift and 4.3-fold quantum yield increase in emission spectrum due to taurodeoxycholate (TDOC) micelle-complex formation. Complex formation also caused a shift in the center of the major lifetime distribution from 11.7 to 15.1 ns, and more than doubled the polarization and anisotropy decay parameters. These data supported an earlier model of colipase-micelle binding that suggested that Tyr-55 was inserted into the interior of the TDOC micelle upon binding (J.C. McIntyre, P. Hundley and W.D. Behnke, Biochem. J. 245 (1987) 821). Identical experiments on a DNS-Tyr-59 derivative of colipase showed that Tyr-59 did not specifically interact with micelles. Moreover, acrylamide quenching data suggest an alteration in the protein environment surrounding DNS-Tyr-59 such that during complex formation, the efficiency of quenching of DNS-Tyr-59 increases.

Acrylamides↗
PubMed1978

Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreases.

1. Colipase was purified from canine pancreatic juice and found to have certain specificity in its reaction with various pancreatic lipases. 2. This colipase will stimulate the lipolytic activities of lipases isolated from canine, bovine and porcine pancreas but not lipases from a fungus, or from human and rat pancreases. 3. Characterization of these lipases showed (a) the molecular dimension of rat lipase is very different from the other lipases; (b) the pIs of canine, porcine and bovine lipases are almost identical but different from the pIs of rat, human and Candida (a fungus) lipases; and (c) the antiserum prepared against canine lipase will also react with lipases from human, hog and cow pancreases but not with rat and Candida lipases. 4. These physical differences can explain partly the difference in reaction between the various lipases and the canine colipase.

Animals↗
PubMed1991

Assignment of the human pancreatic colipase gene to chromosome 6p21.1 to pter.

Pancreatic colipase is a 12-kDa polypeptide cofactor for pancreatic lipase (EC 3.1.1.3), an enzyme essential for the absorption of dietary long-chain triglyceride fatty acids. Colipase is thought to anchor lipase noncovalently to the surface of lipid micelles, counteracting the destabilizing influence of intestinal bile salts. Using primers derived from the known amino acid sequence, we have used the polymerase chain reaction to produce a cDNA clone corresponding to the complete coding region of the human procolipase mRNA. Southern blot analysis of genomic DNA from a panel of mouse-human somatic cell hybrids indicated that the colipase gene (CLPS) resides on human chromosome 6. Further analysis of somatic cell hybrids carrying chromosome 6 translocations permitted regional localization of CLPS to the 6p21.1-pter region.

Blotting, Southern↗
PubMed1999

Colipase: structure and interaction with pancreatic lipase.

Colipase is a small protein cofactor needed by pancreatic lipase for the efficient dietary lipid hydrolysis. It binds to the C-terminal, non-catalytic domain of lipase, thereby stabilising an active conformation and considerably increasing the overall hydrophobic binding site. Structural studies of the complex and of colipase alone have clearly revealed the functionality of its architecture. Interestingly, a structural analogy has recently been discovered between colipase and a domain in a developmental protein (Dickkopf), based on sequence analogy and homology modeling. Whether this structural analogy implies a common function (lipid interaction) remains to be clarified. Structural analogies have also been recognised between the pancreatic lipase C-terminal domain, the N-terminal domains of lipoxygenases and the C-terminal domain of alpha-toxin. These non-catalytic domains in the latter enzymes are important for interaction with membranes. It has not been established if these domains are also involved in eventual protein cofactor binding as is the case for pancreatic lipase.

Amino Acid Sequence↗
PubMed1987

Studies on the effect of bile and lipolysis products on pancreatic lipase and colipase activity in vitro.

This study shows that human bile, at a concentration in the range of that found in the intestinal lumen, inhibited the hydrolysis of emulsified triolein by pancreatic lipase in vitro. The addition of colipase in excess to lipase failed to restore lipolytic activity. In contrast, a partially degraded emulsion of triolein containing diacylglycerol, monoacylglycerol and free fatty acid was hydrolysed by lipase and colipase in the presence of bile. The results presented in this paper allow to conclude that triacylglycerol digestive products associated with emulsified lipid played an important role in the intraluminal degradation of dietary fat by pancreatic lipase and colipase in the presence of bile.

Bile↗
PubMed1997

Neutron crystallographic evidence of lipase-colipase complex activation by a micelle.

The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.

Animals↗