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PubMed1991

Immunochemical determination of porcine pancreatic colipase: differentiation between procolipase and its trypsin-activated form.

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative determination of porcine pancreatic colipase. Calibration curves were established by coating polystyrene immunoplates with pure procolipase or its trypsin-activated derivative. Bound antigen was detected with antiporcine procolipase polyclonal antibodies. Under optimizing conditions, the minimal detectable amount of porcine colipase was 0.1 ng, which is about 1,000 times less than the minimal amount that can be assayed titrimetrically. The useful range of the immunoassay was between 0.1 to 1 ng (2-20 micrograms/L). Under standard assay conditions, no distinction can be made between the precursor and activated forms of the cofactor. Results of immunochemical determinations of colipase in porcine pancreatic juice and tissue extract were in good agreement with those obtained with the potentiometric method. The specific determination of activated colipase in pancreatic juice was performed by coating the immunoplates with antigen in solution in PBS with 0.5 g/L of Tween 20. The detergent selectively impaired the binding of procolipase to the plate. Determination of colipase in human pancreatic juice carried out under the same experimental conditions showed that the minimal amount of human cofactor detectable with ELISA was 1 ng due to partial immunological crossreactivity of the human and porcine proteins. Immunoassay performed with antiporcine procolipase monoclonal antibodies (Mab) showed lower sensitivity than that performed with polyclonal antibodies. However, Mab 72.11, a monoclonal antibody that reacted only with porcine procolipase, allowed specific detection and differential determination of the precursor form of porcine colipase in pancreatic juice. ELISA performed with pure human colipase indicated that no antiporcine procolipase monoclonal antibodies cross-reacted with the human cofactor.

Animals↗
PubMed1977

Lack of adaptation of pancreatic colipase in rats and mice.

A new automated potentiometric method for the determination of colipase was developed, taking advantage of the reactivation of purified lipase, in the presence of bile salt and at pH 6.5. High-fat and high-starch diets induced an opposite regulation of lipase and amylase in the rat pancreas. At the same time, the level of colipase was not influenced by nutrition. During fasting and in alloxan diabetes, the specific activity of lipase almost doubled, that of amylase decreased sharply, and colipase was not affected in the rat pancreas. In obese-hyperglycemic mice, suffering from obesity, hyperinsulinism, and moderate diabetes, there was also no regulation of pancreatic colipase. Thus, at variance with a number of hydrolases, there was no dietary or hormonal adaptation of colipase. However, this was probably without any bearing on intraluminal lipolysis. Indeed, comparison of lipase and colipase activities in pancreas and in small intestine suggests that colipase concentration is not a limiting factor of intraluminal lipolysis. The molecular mechanism of this assumption is discussed on the basis of in vitro studies.

Adaptation, Physiological↗
PubMed1992

Gastric inhibitory polypeptide stimulates pancreatic lipase and colipase synthesis in rats.

Effects of short-term infusion and long-term injection of gastric inhibitory polypeptide (GIP) on changes in pancreatic lipase and colipase contents in rats were studied, and mRNAs encoding for lipase and colipase were determined by Northern blot hybridization with specific cDNA probes. GIP infused at a dose of 3 micrograms/h for 24 h significantly increased the pancreatic lipase content by 34% (P less than 0.05) but had no significant effect on colipase and amylase contents. No change in mRNAs encoding for these proteins was found after infusion of GIP for 24 h. Injection of GIP (5-60 micrograms/kg) three times a day for 5 days dose dependently increased the contents of lipase and colipase, with the increase in colipase being more prominent. Injection of GIP for 5 days at a dose of 30 micrograms.kg-1.day-1 increased colipase and lipase contents by 52 and 25%, and their corresponding mRNAs by 60 and 160%, respectively. The amylase mRNA was not changed by injection of GIP. It is concluded that GIP has a specific stimulatory effect on the synthesis of pancreatic lipase and colipase at both pretranslational and translational levels.

Animals↗
PubMed2000

Parallel secretion of pancreatic phospholipase A(2), phospholipase A(1), lipase, and colipase in children with exocrine pancreatic dysfunction.

The cosecretion of pancreatic lipase and colipase are important in normal fat digestion. As adsorption of phosphatidylcholine to the lipid substrate interferes with lipase activity, hydrolysis to lysophosphatidylcholine with subsequent desorption is also essential for fat digestion. There are some data regarding the secretion of pancreatic phospholipases in normal adults but none in children or patients with pancreatic disease. In the present study, we aimed a) to develop an accurate fast assay method to measure phospholipase A(2) and b) to determine the secretion rate of pancreatic phospholipase A(2) and whether it is cosecreted with lipase and colipase in children with exocrine pancreatic dysfunction. Nine male patients aged 0.5 to 16 y (seven with cystic fibrosis, two with malabsorption) underwent pancreatic stimulation tests. Their colipase and lipase secretion rates were measured by titrimetric methods and phospholipase A(2) and A(1) by phosphorus magnetic resonance spectroscopy ((31)P NMR). It was found that the phospholipases, colipase, and lipase were absent in the two patients with pancreatic insufficiency. In patients with normal absorption, there were marked inter-and intrasubject variations of lipase, colipase, and phospholipase secretion rates that were consistent with the degree of exocrine pancreatic dysfunction. However, in the three 20-min stimulation periods of the pancreatic function test, pancreatic phospholipase is cosecreted with lipase and colipase, and average colipase and phospholipase A(2) secretion rates follow a similar or parallel pattern. These findings are consistent with the important role of pancreatic phospholipases in intestinal phospholipid hydrolysis leading to the desorption of phospholipids from the lipid substrate and enhancing lipid hydrolysis and phospholipid absorption.

Adolescent↗
PubMed1993

Structure of the canine pancreatic colipase gene includes two protein-binding sites in the promoter region.

Characterization of a lambda phage genomic clone, CL5A, which encodes the canine pancreatic colipase gene, revealed the primary structure of 987 nucleotides (nt) of 5'-flanking sequence, 2066 nt defining the primary transcriptional unit, which is organized into three exon sequences, and 130 nt of 3'-flanking sequence. Exon 1 encodes the amino-terminal signal peptide, the propeptide (Val1-Pro-Asp-Pro-Arg), and the hydrophobic lipid-binding region (Gly6-Ile-Ile-Ile) at the amino terminus of the mature coenzyme. Exon 2 encodes carboxylate residues (Glu12 and Glu15) likely to be involved in binding of pancreatic lipase to colipase at the aqueous-lipid interface. Exon 3 encodes the hydrophobic sequence (Leu54-Tyr-Gly-Tyr-Tyr) that is essential for binding the central tightly structured disulfide-bonded region of the coenzyme to lipid. Southern blot analysis was consistent with the presence of a single-copy colipase gene and a potential colipase gene homologue. Among 16 tissues examined by Northern blot analysis, colipase expression was detected only in pancreas. Proteins contained in nuclear extracts prepared from dog pancreas conferred two regions of DNase I protection coincident for both coding and noncoding strands (positions -62 to -44 (CL-I site) and -128 to -106 (CL-II site) in the coding strand). Competition gel mobility shift experiments indicated that protein-DNA interactions that occur at colipase sites I and II are sequence- and protein-specific and unrelated to the PAN-binding sequence described in the 5'-enhancer region of the rat chymotrypsin B gene (Nelson, C., Shen, L.-P., Meister, A., Fodor, E., and Rutter, W. J. (1990) Genes & Dev. 4, 1035-1043). Nuclear extracts from pancreas and brain, but not liver, contain similar CL-I- and CL-II-binding proteins. CL-I and CL-II represent protein-binding elements that may participate as additional promoter regions in regulated expression of the colipase gene. CL-I contains a central homopolymeric d(G) sequence. CL-II shows a GC-rich region on the noncoding strand (5' GGGGGCGTGT 3') that is similar (8/9 match) to the Sp1-binding sequence.

Amino Acid Sequence↗
PubMed2000

Kinetic behavior of the pancreatic lipase-colipase-lipid system.

Pancreatic lipase is a surface-active protein that binds avidly to interfaces comprised of the substrates and products of lipolysis. However, both lipase binding to substrate-containing particles and subsequent interfacial catalysis are inhibited by a number of amphipathic molecules. The most thoroughly studied of these, phosphatidylcholine, is a common constituent of membranes and intestinal lipid contents. Colipase, a surface-active cofactor of lipase, relieves inhibition by phosphatidylcholine in several ways. Through protein-protein interactions, colipase helps anchor lipase to surfaces and stabilizes it in the open conformation. Within the interface, colipase packs more efficiently with substrates and products of lipolysis than with phosphatidylcholine, thereby concentrating these reactants in the vicinity of colipase. This enrichment of lipase substrates and products in the vicinity of colipase enhances lipase-lipid interactions. The result is that colipase facilitates the adsorption of lipase to the interface and, possibly, increases the availability of substrate to the enzyme. Thus, the functional unit in intestinal lipolysis appears to be a lipase-colipase-reactant complex.

Humans↗
PubMed1990

The anticoordinate changes of pancreatic lipase and colipase activity to amylase activity induced by adrenalectomy in normal and diabetic rats.

The effect of adrenalectomy (Adx) on pancreatic lipase and colipase activities in normal, streptozotocin-induced diabetic, and obese Zucker rats was studied. It was found that Adx induced a rapid decrease in amylase activity, a rapid increase in lipase activity, and a slow increase in colipase activity in normal rats. Twenty days after Adx, the levels of amylase activity were 70% lower than control, whereas the activities of lipase and colipase were 55 and 25% higher than control, respectively. The replacement of hydrocortisone normalized the changes in the three enzyme activities. Injection of insulin partially corrected the amylase activity, but had no significant effect on the activities of lipase and colipase. In streptozotocin-induced diabetic rats and obese Zucker rats, amylase activity was decreased, and lipase and colipase activities increased, compared to normal rats. These changes of enzyme activity were further enhanced by Adx. It is concluded that Adx results in an anticoordinate change in pancreatic lipase, colipase, and amylase activities, suggesting that glucocorticoid also influences the synthesis of the pancreatic lipolytic enzymes.

Adrenal Glands↗
PubMed1983

Evolutionary studies on pancreatic colipase.

In this evolutionary study the following criteria have been used to prove the existence of colipase: 1. It restores the activity of human and porcine pancreatic lipase inhibited by bile salt. 2. It cross-reacts with antisera to human and porcine colipases. 3. Its restoration of lipase activity, inhibited by bile salt, in the tributyrin assay system, is prevented by antiserum to colipase. 4. It is a heat-stable, low-molecular-weight protein (molecular weight about 10 000 by gel-filtration). The occurrence of colipase has been verified in the exocrine pancreatic cells from hagfish (Myxine glutinosa), ratfish (Chimaera monstrosa), rayfish (Raja radiata). Greenland shark (Somnius microcephalus) and dogfish (Squalus acanthius). No colipase activity could be found in the gastric juice of crayfish (Pacifastacus leniusculus). These results indicate that colipase envolved in the vertebrates before the organized exocrine pancreatic gland and occurred simultaneously with the bile salts/bile alcohols.

Animals↗
PubMed1982

Detection of colipase in serum and urine of pancreatitis patients.

Colipase, like other pancreatic proteins, is liberated into the circulation in acute pancreatitis. Its concentration was measured in serum by a turbidimetric and in urine by a titrimetric method. The principle of both assays is based on the reactivation of bile acid inhibited, pure human pancreatic lipase by colipase. Whereas in healthy individuals colipase was found neither in serum nor urine (detection limit approximately 6.5 micrograms/1), a wide concentration range was observed in 29 patients with acute pancreatitis. Urine values varied between 3.8 and 7121 micrograms colipase/g creatinine; in serum levels up to 664 micrograms/1 were found. There was no correlation with serum lipase activity: On a molar basis, the ratio of serum colipase to serum lipase ranged between less than 0.04 and 2.14, but was below 1 in most sera. Colipase is rapidly removed from the circulation by glomerular filtration, its elimination rate from serum being more than twice as fast as that of lipase. This results in a constant decrease of the colipase/lipase ratio during the course of the disease. Probably determination of colipase is of no direct diagnostic value in pancreatic disorders, but our findings are of considerable significance for the measurement of serum lipase in the presence of bile acids, particularly with regard to turbidimetric assays. We conclude that lipase activity values obtained by these methods are mainly dependent on the degree of saturation of the enzyme with its cofactor and not on the true lipase concentration.

Amylases↗
PubMed1987

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase.

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.

Animals↗
PubMed1981

Horse pancreatic lipase. Interaction with colipase from various species.

Horse pancreatic lipase has been purified from tissue homogenates. Molecular and catalytic properties of horse lipase are comparable to those of the pancreatic lipases previously isolated. Kinetic studies of the inhibition of horse lipase activity by bile salts and of reactivation by pure colipase from three species (horse, ox and pig) allowed to calculate the apparent dissociation constant (Kd) of the lipase-colipase complex in the presence of the substrate (triolein). Identical values of Kd were found in all three cases (Kd = 1.1 10(-9) M). These values are lower by several orders of magnitude than that published for the binding between lipase and colipase in the absence of substrate. Qualitative experiments show that the activation of horse lipase can be accomplished by rat, dog and chicken colipase as well. The interaction between lipase and colipase is enhanced when the complex is adsorbed at the lipid-water interface. This specific protein-protein interaction is preserved in heterologous mixtures using colipases from other animal species.

Animals↗
PubMed1992

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.

Amino Acid Sequence↗
PubMed1979

High-resolution proton magnetic resonance study of porcine colipase and its interactions with taurodeoxycholate.

A high-resolution 270-MHz proton NMR study of procine colipase I has been performed, and the resonances in the aromatic region of the spectrum have been assigned to amino acid residues by pH titration and decoupling experiments. The apparent pKa values of the three tyrosines were calculated to be 10.2, 10.3, and 11.8 with one of the tyrosines having properties of a "buried" residue. A tentative assignment to the amino acid residues in the primary seuqence of colipase will be discussed. The effects of taurodeoxycholate (TDC) and a positively charged deoxycholate derivative on the aromatic region of the colipase NMR spectrum indicate that all tyrosines and one histidine are affected by the bile-salt binding, suggesting that the TDC molecules bind near these residues to a hydrophobic region on colipase. Measurements and calculations on the line width of the C(18) methyl group resonance suggest that the line-width increase of this resonance upon interaction of TDC with colipase to a large extent can be explained as due to the slower tumbling of the TDC molecules bound to colipase.

Amino Acid Sequence↗
PubMed1987

The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.

Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52.

Acrylamide↗
PubMed1998

Ion pairing between lipase and colipase plays a critical role in catalysis.

Among the polar interactions occurring in pancreatic lipase/colipase binding, only one ion pair involving lysine 400 on lipase and glutamic acid 45 on colipase has been described. These residues are strictly conserved among species, suggesting that the ion pair is likely to play an important role. Therefore, in order to prevent this interaction, mutations intended to neutralize or inverse the charge of these residues have been introduced in the cDNAs encoding horse lipase and colipase. The recombinant proteins have been expressed in insect cells, and their catalytic properties have been investigated. In all cases, preventing the formation of the correct ion pair Lys400/Glu45 leads to lipase-colipase complexes of reduced affinity unable to perform an efficient catalysis, notably in the presence of bile salt micelles. Diethyl p-nitrophenyl phosphate inhibition experiments with either mutant lipase or mutant colipase indicate a poor stabilization of the lipase flap. These results suggest that the ion pair plays a critical role in the active conformation of the lipase-colipase-micelle ternary complex by contributing to a correct orientation of colipase relative to lipase resulting in a proper opening of the flap.

Animals↗
PubMed1992

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.

Amino Acid Sequence↗
PubMed1992

Evidence of a stimulatory effect of cyclic AMP on pancreatic lipase and colipase synthesis in rats.

The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of 35S-cystine. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cystine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 microM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2 h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat.

Amylases↗
PubMed1992

Enzyme substitution in pancreatic disease: is colipase activity sufficient?

The aim of the present study was to determine colipase activity in various pancreatic enzyme tablets to ascertain whether these contained sufficient amounts of colipase to activate lipase during fat digestion. Colipase activity in all preparations tested exceeded that of lipase activity by a factor of 1.4-1.9 on a molar activity basis. Since optimal activity of lipase is obtained with colipase being present in a colipase to lipase molar activity ratio of 1.0, it is concluded that these preparations contain a sufficient amount of colipase to activate lipase.

Capsules↗