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PubMed1991

How to protect human pancreatic enzyme activities in frozen duodenal juice.

We determined whether activity of pancreatic enzymes could be maintained in frozen duodenal juice by diluting the specimens or by adding nutrients or a chymotrypsin inhibitor. Human duodenal juice was obtained during cholecystokinin octapeptide IV administration. Trypsin, chymotrypsin, lipolytic, lipase, and colipase activities were measured in fresh undiluted or diluted (1:4 and 1:16 with saline and T-tube bile) duodenal juice as well as after adding CaCl2, casein, triolein, or a chymotrypsin inhibitor. Subsequently, the samples were frozen at -20 degrees C, and enzyme activities were measured at 1, 2, 3, 7, 14, 28, and 56 days. Activities of chymotrypsin and colipase did not change during freezer storage. Trypsin survival was variable in juice from different subjects. By contrast, in duodenal juice to which no nutrient or only CaCl2 had been added, 90%, 65%, and 40% (P = 0.05 vs. undiluted) of lipolytic activity was lost by 56 days in undiluted and 1:4 or 1:16 diluted duodenal juice samples, respectively. The loss of lipolytic activity was prevented (P less than 0.05) by adding casein or casein and triolein to undiluted and 1:4 diluted samples and turkey egg white to undiluted samples. The loss of lipolytic activity was strongly associated with loss of lipase activity (r = 0.97) but only weakly associated with loss of colipase activity (r = 0.49). In summary, chymotrypsin and colipase are well preserved in frozen duodenal juice and can be used to accurately assess concentrations of pancreatic enzymes after thawing frozen duodenal samples. If it is necessary to measure lipolytic activity after freezing samples, lipase can be maintained by adding casein or a chymotrypsin inhibitor to juice before freezing.

Colipases↗
PubMed2004

Human pancreatic lipase-related protein 2 is a galactolipase.

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

Animals↗
PubMed1994

Pancreatic digestive hydrolase activities in growing rats fed alternately on raw and heated soya-bean flour.

The time-course effects of two diets containing raw (RSF) or heated (HSF) soya-bean flour on the digestive enzyme levels in the pancreas and in pancreatic juice were investigated in growing rats fed, alternately, on RSF or HSF diets for two 4-week periods. These values were compared with those obtained in a control group fed on a casein diet. RSF and HSF diets lowered N balance (84.8 (SE 0.9), 82.6 (SE 0.8) and 79.9 (SE 0.8)% with control, HSF and RSF diets respectively, at the third week). However, they increased protease activities compared with the control diet (3-fold for trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) activities in pancreas contents and outputs with the RSF diet; 2-fold for trypsin in pancreas contents and outputs and by 60% for chymotrypsin contents with the HSF diet). The poorer nutritional N utilization might be attributable to soya-bean flour heat-stable (lectins) and heat-labile components (trypsin inhibitors). The decrease in lipid apparent digestibilities in RSF and HSF diets (97.0 (SE 0.8), 91.1 (SE 0.9) and 90.4 (SE 0.7)% with control, RSF and HSF diets at the seventh week) were correlated with a diminution in apparent lipase (EC 3.1.1.3; measured without addition of exogenous colipase), potential lipase (measured with addition of saturated amounts of exogenous colipase) and colipase activities. Compared with control values, gains in potential and apparent lipase outputs were diminished by nearly 40% and gain in colipase outputs by 60% with RSF and HSF diets. These results show clearly that heated or raw soya-bean flours have a significant inhibitory effect on lipase digestive enzyme activities in the pancreas and in its secretion, which might explain impaired lipid digestibility.

Animals↗
PubMed1998

Pancreatic lipase-related protein type I: a specialized lipase or an inactive enzyme.

The existence of pancreatic lipase-related protein 1 (PLRP1) in vertebrates has been postulated based on the screening of pancreatic cDNA libraries from different species. In this paper, we report the presence of variable amounts of PLRP1 relative to colipase-dependent lipase (PL) in adults from several species. Only a very low lipase activity could be detected for native or recombinant PLRP1 using a large variety of substrates and conditions. Interestingly, this activity is dependent on the presence of bile salts and colipase and PLRP1 is shown to possess the same affinity as PL for colipase. Modelling investigations revealed some interesting differences between PLRP1 and PL, notably concerning substitutions in the C-terminal domain which might affect the bending motion of this domain relative to the N-terminal domain in PLRP1. The potential impact of these differences on the lack of lipase activity of PLRP1 was investigated using chimeric proteins designed by C-terminal domain exchange between dog PLRP1 and horse PL. Analysis of the catalytic properties of the chimera clearly indicated that the C-terminal domain exchange neither inactivates the horse enzyme nor results in an active dog PLRP1. From these findings, it can be concluded that the PLRP1 C-terminal domain is fully functional with respect to colipase binding. The lack of lipase activity or the still undetermined function of PLRP1 is likely to result mainly from particular features of the N-terminal domain.

Amino Acid Sequence↗
PubMed2003

Effect of long-term high-fat feeding on the expression of pancreatic lipases and adipose tissue uncoupling proteins in mice.

INTRODUCTION: A diet containing a high amount of fat has been shown, in short-term studies, to increase the expression of pancreatic lipase and colipase. AIM: To investigate the effects of long-term high-fat-feeding (113 days) on the mRNA expression of pancreatic lipase, colipase, pancreatic lipase-related proteins (1 and 2), and uncoupling proteins during the development of obesity and glucose intolerance. METHODOLOGY: Mice were fed either a high-fat or standard diet and killed after 3, 13, 57, and 113 days. Brown and white adipose tissue and pancreas were collected for mRNA extraction [corrected]. RESULTS: The high-fat-fed mice became obese and glucose-intolerant by 113 days. The high-fat diet increased lipase (p < 0.05) expression initially. At the end of the experiment, the lipase levels had decreased to the level of the control. Colipase levels did not change during the first 57 days of high-fat feeding but decreased below control levels by 113 days (p < 0.05). The high-fat diet increased brown adipose tissue uncoupling protein 1 (UCP1)(p < 0.005) expression but not the expression of uncoupling protein 2. CONCLUSION: Long-term high-fat feeding, leading to glucose intolerance, occurs with a simultaneous decrease in the mRNA expression of pancreatic lipase and colipase and an increase in UCP1 expression.

Adipose Tissue↗
PubMed1982

Response of the exocrine pancreas to quantitative and qualitative variations in dietary lipids.

In the rat, pancreatic amylase and, to a lesser extent, lipase adapt quantitatively to the amount of their respective substrates in the diet by an increase in specific activity and total contents (range of variation, fivefold for amylase and twofold for lipase). Colipase responded to protein intake (r = 0.85, P less than 0.01) and not to lipids provided protein intake was below 3.5 g or above 6.0 g. With this latter amount of protein, a maximal level was obtained, even with 2% lipid in the diet. Between 3.5 and 6.0 g, lipid intake was found to modulate colipase in parallel with lipase. When different types of fat were compared, the degree of saturation was found to have no impact on lipase, colipase, and amylase. Diets containing medium-chain triglycerides (C8-C10) did not maximally increase specific activity and total content of lipase and colipase, whereas they did not repress amylase as much longer chain triglycerides did. With coconut oil (45% C12), lipase was maximally stimulated but amylase was not maximally repressed, showing that the regulation of the hydrolases may be partly reciprocal and partly independent.

Amylases↗
PubMed1995

In vitro rat pancreatic digestive enzyme activities and raw and heated glandless cottonseed and soybean flours.

Higher nitrogen and lipid digestibilities have been obtained with diets containing cottonseed flour rather than soybean flour. To explain these results, in vitro studies were carried out to compare the effects of raw and heated glandless (without gossypol) cottonseed flours versus soybean flours on pancreatic digestive enzyme activities. These effects were compared with those obtained without addition of flour in standard assays. Apparent lipase (lipase colipase dependent) and potential lipase (lipase with saturating amounts of colipase), colipase, phospholipase A2, amylase, trypsin and chymotrypsin activities were measured on specific substrates. Phospholipase A2 and amylase activities were enhanced, while chymotrypsin activity was diminished with both raw and heated flours. Compared with raw and heated soybean flours, raw and heated cottonseed flours promoted higher potential lipase, chymotrypsin, trypsin and lipase activities. Heat treatment of cottonseed flour enhanced apparent lipase, colipase, chymotrypsin, trypsin activities and diminished potential lipase, phospholipase A2 and amylase activities. When soybean flour was heated, apparent lipase, phospholipase A2, chymotrypsin, trypsin and amylase activities were raised while those of potential lipase were decreased. Our findings show that in vitro raw or heated cottonseed flours affect less digestive enzymes than raw or heated soybean flours, apparent lipase activity excepted. Moreover, only chymotrypsin activities were seriously lowered with both flours, especially with raw soybean flour. Hypotheses are suggested to account for the differences in alterations.

Amylases↗
PubMed1989

Hydrolysis of triacylglycerol arachidonic and linoleic acid ester bonds by human pancreatic lipase and carboxyl ester lipase.

The hydrolysis of polyenoic fatty acid ester bonds with pure human colipase-dependent lipase, with carboxyl ester lipase (CEL) and with these enzymes in combination was studied, using [3H]arachidonic- and [14C]linoleic acid-labelled rat chylomicrons as a model substrate. During the hydrolysis with colipase-dependent lipase, the amount of 3H appearing in 1,2-X-diacylglycerol (DG) markedly exceeded that of 14C. When CEL was added in addition this [3H]DG was efficiently hydrolyzed. CEL alone hydrolyzed the triacylglycerol (TG) at a low rate. The hydrolysis pattern with human duodenal content was similar to that seen with colipase-dependent lipase and CEL in combination. Increasing the concentration of taurodeoxycholate (TDC) and taurocholate (TC) or of TDC alone stimulated the hydrolysis of [3H]- and [14C]TG, but increased the accumulation of labelled DG that could act as substrate for CEL. It is suggested that very-long-chain polyenoic fatty acids of DG formed during the action of the colipase-dependent lipase on TG containing these fatty acids may be a physiological substrate for CEL.

Arachidonic Acid↗
PubMed2000

Isolation of a funnel-web spider polypeptide with homology to mamba intestinal toxin 1 and the embryonic head inducer Dickkopf-1.

We have isolated and determined the amino acid sequence of a novel peptide component from the venom of the Australian funnel-web spider Hadronyche versuta. This 68-residue toxin, ACTX-Hvf17, does not function like classical neurotoxins in modulating ion channel function as evidenced by its lack of insecticidal activity and its inability to affect vertebrate smooth or skeletal muscle contractility. The peptide shows significant sequence homology with mamba intestinal toxin 1 (MIT1) and to a lesser extent with a variety of colipases. The strong structural homology between MIT1 and porcine colipase leads us to propose that ACTX-Hvf17 also adopts the MIT1/colipase three-dimensional fold. However, we show that ACTX-Hvf17 has no colipase activity and does not stimulate muscle contractility like MITI. We also show that MIT1 and ACTX-Hvf17 display significant sequence homology with the C-terminal cysteine-rich domain of the Dickkopf-1 family of proteins that induce head formation in developing embryos, which leads us to propose that this domain of Dickkopf-1 also adopts the MIT1 colipase fold.

Amino Acid Sequence↗
PubMed2005

Role of the lid hydrophobicity pattern in pancreatic lipase activity.

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240-252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile salt-coated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240-252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.

Algorithms↗
PubMed1990

Altered synthesis of some secretory proteins in pancreatic lobules isolated from streptozotocin-induced diabetic rats.

The in vitro incorporation of [35S]cysteine into lipase, colipase, amylase, procarboxypeptidase A and B, and the serine proteases and total proteins was studied in pancreatic lobules isolated from normal and diabetic rats with or without insulin treatment. The incorporation of [35S]cysteine into total proteins was 65% greater in pancreatic lobules from diabetic animals than from normal rats. The increased incorporation was partly reversed by insulin treatment (2 U/100 g/day for 5 days) of diabetic rats. The relative rates of biosynthesis for amylase and the procarboxypeptidases in diabetic pancreatic lobules were decreased by 75 and 25%, respectively, after 1 h of incubation, while those for lipase, colipase, and the serine proteases were increased by 90, 85, and 35%, respectively. The absolute rates of synthesis for these enzymes changed in the same direction as the relative rates in diabetic lobules, except that for the procarboxypeptidases, which did not change. The changed rates of biosynthesis for the pancreatic enzymes were reversed by insulin treatment of the diabetic rats. Kinetic studies showed that the incorporation of [35S]cysteine into amylase, lipase, and colipase was linear until up to 2 h of incubation in normal pancreatic lobules, while in the diabetic lobules the incorporation into lipase and colipase was accelerated, reaching a plateau level already after 1 h of incubation. It is concluded that the biosynthesis of pancreatic secretory proteins in diabetic rats is greatly changed both in terms of quantity and kinetics.

Animals↗
PubMed2005

Peripheral protein adsorption to lipid-water interfaces: the free area theory.

In fluid monolayers approaching collapse, phospholipids and their complexes with diacylglycerols hinder adsorption to the monolayer of the amphipathic protein, colipase. Herein, a statistical, free-area model, analogous to that used to analyze two-dimensional lipid diffusion, is developed to describe regulation by lipids of the initial rate of protein adsorption from the bulk aqueous phase to the lipid-water interface. It is successfully applied to rate data for colipase adsorption to phospholipid alone and yields realistic values of the two model parameters; the phospholipid excluded area and the critical free surface area required to initiate adsorption. The model is further developed and applied to analyze colipase adsorption rates to mixed monolayers of phospholipid and phospholipid-diacylglycerol complexes. The results are consistent with complexes being stably associated over the physiologically relevant range of lipid packing densities and being randomly distributed with uncomplexed phospholipid molecules. Thus, complexes should form in fluid regions of cellular membranes at sites of diacylglycerol generation. If so, by analogy with the behavior of colipase, increasing diacylglycerol may not trigger translocation of some amphipathic peripheral proteins until its abundance locally exceeds its mole fraction in complexes with membrane phospholipids.

Adsorption↗
PubMed2000

The open lid mediates pancreatic lipase function.

Pancreatic triglyceride lipase (PTL) and the homologous pancreatic lipase related protein 2 (PLRP2) provide a unique opportunity to understand the molecular mechanism of lipolysis. They differ in substrate specificity, sensitivity to bile salts, and colipase dependence despite their close amino acid and tertiary structure identity. One important structure, present in both lipases, is the lid which occupies different positions in the inactive and active forms of PTL. We investigated the role of the lid in lipase function by site-specific mutagenesis. By exchanging the lids between PTL and PLRP2, we created two chimeric lipases. Additionally, we made multiple substitution mutations in the PTL lid. PLRP2 with the PTL lid had kinetic properties similar to PLRP2. PTL with the PLRP2 lid was greatly impaired and had no activity at micellar bile salt concentrations even in the presence of colipase. Both chimeras showed interfacial activation suggesting that the closed lid position was maintained. A series of substitution mutations were made in positions Arg257 and Asp258. These mutations demonstrated the importance of these two residues to maintaining the normal activity, triglyceride acyl chain specificity, and colipase interaction of PTL. The preserved interfacial activation in the chimeras, the similar crystal structure of the two lids in the closed position, and the importance of Arg257 and Asp258 in mediating the open conformation of the lid argue that the position of the open lid influences the differences in activity against triglycerides, in sensitivity to bile salts, and in colipase dependence between PTL and PLRP2.

Amino Acid Sequence↗
PubMed1984

Inhibition of pancreatic and microbial lipases by proteins.

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.

Animals↗
PubMed1984

Evidence for the existence of procolipase in chicken pancreas and pancreatic juice.

Purified antibodies raised against chicken colipase were coupled to Sepharose 4B and colipase was isolated in a single step by immunoaffinity chromatography from an extract of chicken pancreas prepared under conditions where trypsin activation is avoided. The purified protein has a single amino terminal residue of alanine and its biochemical properties are similar to those of the precursor form of colipase (procolipase) previously isolated from porcine and equine pancreas or pancreatic juice. Further evidence for the existence of procolipase was obtained from kinetic studies of the hydrolysis of the Intralipid emulsion by untreated and trypsin-treated chicken pancreatic juice.

Animals↗
PubMed2006

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase.

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.

Amino Acid Sequence↗
PubMed2004

Rapid exchange of pancreatic lipase between triacylglycerol droplets.

Two types of experiments were performed to study the reversibility of interfacial adsorption of pancreatic lipase (PL) to fat droplets during lipolysis. Lipolysis was measured in olive oil/gum arabic emulsions containing radiolabeled triolein in the presence of bile salts and lecithin at rate-limiting concentrations of porcine PL (PPL) or human PL (HPL). The lipolysis rate in a labeled emulsion, i.e. release of [(14)C]oleic acid, was immediately reduced by around 50% upon dilution with an equal amount of an unlabeled emulsion. Further, lipolysis was rapidly and completely suppressed when a non-exchanging lipase inhibitor was present in the second emulsion. These results indicate hopping of lipase between emulsion droplets. Alternative explanations were excluded. Hopping of PL between triolein droplets stabilized with gum arabic at supramicellar bile salt concentrations was observed only in the presence, not in the absence, of lecithin. Displacement from a trioctanoin-water interface of active HPL by an inactive mutant (S152G) was studied in the presence of bile salts by measuring HPL distribution between the water phase and the oil-water interface. Colipase was limiting for HPL binding to the oil-water interface (colipase to lipase molar ratio: 0.5) and, thus, for lipolysis. Upon adding S152G, which has the same affinity for colipase, inactive and active HPL were found to compete for binding at the oil-water interface. When equal amounts of HPL and HPL S152G were used, the lipolysis rate dropped to half the maximum rate recorded with HPL alone, suggesting that half the active HPL was rapidly desorbed from the oil-water interface. Therefore, under various conditions, PL does not remain irreversibly adsorbed to the oil-water interface, but can exchange rapidly between oil droplets, via an equilibrium between soluble and lipid-bound PL.

Animals↗
PubMed1998

Rat gastric procolipase: sequence, expression, and secretion during high-fat feeding.

BACKGROUND & AIMS: Procolipase, the cofactor for pancreatic lipase, was recently found in the rat stomach using immunohistochemistry. The aim of this study was to determine the sequence of rat gastric procolipase, to evaluate the expression and secretion during high-fat feeding, and to find out the conditions for activation of gastric procolipase to form colipase and enterostatin. METHODS: Gastric procolipase was cloned from a rat complimentary DNA (cDNA) library using a 32P-labeled pancreatic procolipase probe for screening. For the expression of gastric procolipase, rats were fed a high-fat diet for 0, 1, 2, 5, and 14 days. Gastric mucosa was collected for isolation of RNA and gastric juice for measurement of procolipase. After treatment with pepsin, HCl, and trypsin, gastric juice was analyzed on high-performance liquid chromatography for identification of enterostatin. RESULTS: The cDNA sequence for gastric procolipase was identical to that of pancreatic procolipase. High-fat diet decreased the expression of gastric procolipase. Enterostatin was present in the gastric juice, with pepsin and acid involved in the cleavage of gastric procolipase. CONCLUSIONS: Gastric procolipase is activated to release colipase and enterostatin. The role of gastric colipase may be to prepare lipase-catalyzed fat digestion already in the stomach. Gastric enterostatin may be involved in the onset of early satiety.

Amino Acid Sequence↗