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PubMed1989

Cloning and characterization of human pancreatic lipase cDNA.

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.

Amino Acid Sequence↗
PubMed1984

[Catalytic activity of serum lipase during continuous temperature- dependent titrimetry].

The catalytic activity of the pancreatic lipase in serum was measured between 20 degrees C and 37 degrees C by the continuous titrimetric assay. As the temperature was increased, the time course of substrate hydrolysis showed an increasing tendency to become non-linear. The degree of non-linearity and its time of onset depended on the sample, the volume of serum in the assay, and the composition of the triglyceride substrate mixture. Twenty seven series of investigations between 20 degrees C and 32 degrees C (maximal) showed a Q10 value of 1.45 (s = 0.03) and an activation energy of mu = 27.42 +/- 0.565 kJ/mol (6550 +/- 135 cal/mol; mean +/- s). At low assay temperature, the addition of colipase caused a slight deterioration of the reaction kinetics, whereas at higher temperatures it caused a marked improvement in the time course of substrate hydrolysis. We were unable, however, to identify a fixed colipase concentration that would promote a linear reaction irrespective of sample origin and volume. These results show that the assay should be performed at 25 degrees C, and that the test conditions described in 1969 and 1982 are valid without modification. If these conditions are observed, there is no experimental basis for the addition of colipase of the quality commercially available at present.

Catalysis↗
PubMed1994

[Titrimetric measurement of catalytic concentration of pancreatic lipase: state of the art].

Most lipase routine assays are carried out using either soluble substrates or emulsified substrates (triglycerides or olive oil) at low concentrations. Many of these techniques require a secondary standard, which must be titrated beforehand; the need for a reference method is thus compelling. Titrimetric assays have several advantages such as the possibility of employing high substrate concentrations allowing the direct determination of the product of lipolysis in the absence of interfering phenomena. In a recent study it was demonstrated that human lipase activity depends on the zeta-potential of the lipid droplets, the number of hydroxy groups present in each individual bile salt, the aggregation number and the conjugation of bile salts with taurine or glycine. Hydroxypropyl methylcellulose proposed by Tietz et al is to be preferred to gum arabic for being a pure, well defined emulsifier. Ultrasonic homogenizers enable volumes of oil-in-water emulsions, characterized by fine lipid droplets with good homogeneity to be obtained without overheating. Lipolytic activity is completely inhibited by 70 mmol/l of bile salt (regardless of the type) in the absence of colipase. Variable concentrations of colipase are needed to restore the lipase activity in the presence of different bile salts: optimal cofactor concentrations vary from 0.1 mg/l with deoxycholate or cholate to 6 mg/l with taurocholate or glycocholate. Even after optimization of the medium with colipase, marked differences in enzyme activity are noted depending on the bile salt used.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗
PubMed1978

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts.

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.

Animals↗
PubMed1991

Enterostatin suppresses food intake following injection into the third ventricle of rats.

The effect on food intake of an activation peptide from pancreatic pro-colipase, called enterostatin, has been studied after parenteral or third ventricular administration. The activation peptide (enterostatin = Val-Pro-Asp-Pro-Arg = VPDPR) reduced food intake when given intraperitoneally. Low doses of this peptide also reduced food intake when given into the third ventricle, but high doses were ineffective. Enterostatin did not modify the stimulatory effects on food intake of clonidine, an alpha 2-adrenergic agonist, suggesting that its anorectic effects are not mediated via the alpha 2-adrenergic system. These data suggest that enterostatin, an activation peptide from pro-colipase, may play a role in producing satiety.

Analysis of Variance↗
PubMed1985

Trypsin activation of porcine procolipase. Kinetics of activation and effects on lipid binding.

The kinetics of trypsin activation of pancreatic procolipase was investigated and the pH dependence of the binding of procolipase and colipase to a tributyrine-bile salt interface studied. The Km was 0.06 mM and kcat 8 s-1, and was of the same order of magnitude as for the activation of pancreatic zymogens. At basic pH values colipase had a higher affinity for the tributyrine-bile salt interface as compared to procolipase. The trypsin activation of procolipase ensures a rapid degradation of dietary lipids in the intestine.

Animals↗
PubMed1991

Antigen specificity and cross-species reactivity of a monoclonal antibody (mAb 72.11) against porcine pancreatic procolipase.

We have studied the antigen specificity and cross-reactivity of a monoclonal antibody (mAb 72.11) of subclass IgG1, raised against the precursor form of porcine colipase (procolipase), whose epitope lies near the amino terminal region of the polypeptide. mAb 72.11 cross-reacts with native porcine, equine and human procolipase, as shown by immuno-inactivation and ELISA titration studies carried out on pure proteins, pancreatic tissue homogenate or pancreatic juice. The epitope site recognized by mAb 72.11 was further characterized by studying antibody binding to denatured procolipase. Reduced carboxymethylated procolipase reacted with mAb 72.11 in ELISA. Heat inactivated or reduced carboxymethylated porcine procolipase displaced antigen from the complex formed between antibody and native procolipase. The lack of sensitivity of epitope recognized by mAb 72.11 on procolipase to heat denaturation or reduction of the disulfide bridges is indicative that antigen specificity of mAb 72.11 is not dependent on the conformation of the antigenic site. Cross-reactivity of mAb 72.11 with procolipase from the three species demonstrates that substitution of amino acid at positions 1 and 3 causes no loss of antigenicity. Finally, mAb 72.11 was coupled to sepharose to isolate human procolipase from human pancreatic juice and to separate the precursor form from activated colipase non-adsorbed on the column.

Amino Acid Sequence↗
PubMed2002

Transfer of orlistat through oil-water interfaces.

The transfer of radiolabelled orlistat ([14C]orlistat), a potent gastrointestinal lipase inhibitor, through an oil-water interface from a single oil droplet to an aqueous phase was investigated, using an oil drop tensiometer. The absolute transfer fluxes were found to be very low, even in the presence of micellar concentrations of bile salts, which increased their values from 0.2 to 2.5 and 6.5 pmol cm(-2) min(-1) in the presence of 0, 4 and 15 mM NaTDC, respectively. Adding either a lipid emulsion or pure human pancreatic lipase (HPL) or human serum albumin or beta-lactoglobulin had no effect on the flux of transfer of orlistat. The presence of colipase or a mixture of colipase and HPL was found, however, to reduce the flux of orlistat transfer, probably because it partly covered the single oil drop surface, even in the presence of bile salts. Using a finely emulsified system, we investigated the partitioning of orlistat between the aqueous and oil phases, in the absence or presence of bile salts above their CMC (4 mM NaTDC, final concentration). Under these emulsified conditions, orlistat was found to be mostly associated with the oil phase, since more than 98.8% of the total radioactivity was recovered after decantation with the oil phase. The low transfer rates of orlistat, as well as its partitioning coefficient between the oil and the aqueous phases, should help us to better understand the inhibitory effects of orlistat on lipid digestion in humans.

Animals↗
PubMed1989

Interface-mediated inactivation of pancreatic lipase by a water-reactive compound: 2-sulfobenzoic cyclic anhydride.

2-Sulfobenzoic cyclic anhydride (SBA) rapidly and selectively inactivates porcine pancreatic lipase (PPL) only when added during the hydrolysis of an emulsified ester such as tributyrin or dodecyl acetate. The present data suggest that the inactivation of PPL occurs preferentially at the oil/water interface and not in the aqueous phase, since colipase and bile salt were found to adversely affect the inhibition process. Moreover, it is shown that at a molar ratio of SBA to pure PPL of 1, 40% of the lipase activity was already irreversibly lost. Complete inactivation was observed at SBA to pure PPL molar ratios of 120. A 60% inactivation occurred when 0.5 mol of 3H-labeled SBA was attached per mole of PPL. The SBA-inactivated PPL competes for binding to the dodecyl acetate/water interface as efficiently as the native enzyme. Larger SBA concentrations are required when crude lipase preparations are used as well as with pure PPL in the presence of bile salts and colipase. Lipases were found to have variable sensitivities to SBA inactivation, depending on their origin. In the presence of bile salts and tributyrin at pH 6.0, human gastric lipase activity was not affected by the presence of a 10(6) molar excess of SBA.

Animals↗
PubMed1997

Pancreatic lipase structure-function relationships by domain exchange.

We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.

Amino Acid Sequence↗
PubMed1993

Procolipase mRNA: tissue localization and effects of diet and adrenalectomy.

Northern blot analysis has identified procolipase mRNA in rat pancreas, stomach and duodenum. Pancreatic colipase mRNA was increased by high-fat diets. Adrenalectomy increased pancreatic procolipase mRNA, an effect enhanced by high-fat diets. The results suggest that colipase is not unique to the pancreas and that diet and glucocorticoids interact in regulating the transcription of its gene.

Actins↗
PubMed2000

Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering.

In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.

Animals↗
PubMed1996

Exocrine pancreatic secretions in growing pigs fed diets containing fish oil, rapeseed oil or coconut oil.

Two experiments were performed to study the effect of feeding diets containing oils with different fatty acid composition on exocrine pancreatic secretions in growing pigs using two different methods to collect pancreatic juice. In the first experiment, three barrows (initial weight 37 kg) were fitted with a pancreatic pouch re-entrant cannula. An isolated pouch was prepared where the pancreatic duct enters the duodenum. In the second experiment, also using three barrows (initial weight 32 kg), a catheter was inserted into the pancreatic duct. Three wheat starch and fish meal-based diets were formulated to contain either 15 g fish oil, rapeseed oil or coconut oil/100 g. In both experiments, the diets were fed according to a 3 times 3 Latin square design. The volume of pancreatic juice secreted, pH and secretion of bicarbonate, protein, amylase, trypsin, lipase and colipase were not significantly affected by the diets in the first experiment. In the second experiment, chymotrypsin secretion was significantly greater in pigs fed the coconut oil diet, and secretion of carboxyl ester hydrolase was significantly higher in pigs fed the fish oil diet. When compared qualitatively, pigs in Experiment 2 secreted more pancreatic juice; the pancreatic juice had a higher pH, and trypsin, carboxyl ester hydrolase and colipase secretions were substantially higher whereas amylase secretion was lower than for pigs in Experiment 1. The fatty acid composition of the different oils had minor effects on exocrine pancreatic secretion in growing pigs. However, there were considerable differences between the two surgical methods used to collect pancreatic juice, and these differences may be explained by physiological changes induced by the two methods.

Amylases↗
PubMed1991

cDNA sequence and deduced amino acid sequence of human preprocolipase.

Complementary DNA clones for human pancreatic colipase were identified in human pancreatic cDNA libraries by hybridization with a pool of synthetic oligonucleotides containing all possible coding sequences for amino acids 75 to 80 of the partial human colipase protein sequence (Sternby, et al. Biochim Biophys Acta 1984;784:75). Alignment of overlapping cDNA clones yielded an mRNA sequence of 504 nucleotides [not including the poly(A) tail] encoding a polypeptide of 112 amino acids. The prepeptide comprised 17 amino acids, with an amino-terminal cluster of charged residues followed by a hydrophobic core of 12 residues typical of leader sequences. The deduced human procolipase sequence comprised 95 residues, including a propeptide of 5 residues. It was in complete agreement with the partial sequence previously obtained by protein sequencing. Northern blot analysis revealed that the polyadenylated preprocolipase transcript had a length of approximately 680 nucleotides.

Amino Acid Sequence↗
PubMed1993

Identification of enterostatin, the pancreatic procolipase activation peptide in the intestine of rat: effect of CCK-8 and high-fat feeding.

Enterostatin, the procolipase activation peptide, has been suggested in previous studies to act as a satiety signal for food intake, with a specificity for fat intake. In this study, by use of a competitive enzyme-linked immunosorbent assay with a detection limit of 4.115 nmol/L and within 6% intra- and interassay variation, the immunoreactive and chromatographic characterization of enterostatin in intestinal content was undertaken in Sprague-Dawley rats. Following intravenous infusion of cholecystokinin octapeptide (CCK-8; 200 pmol/kg/h) for 60 min, the concentration of intestinal enterostatin increased from a basal level of 2.0 +/- 0.7 microM to 5.64 +/- 1.1 microM at time point 60 min. The enterostatin level remained at 4.24 +/- 0.54 microM for 120 min after the CCK infusion had ceased. Pancreatic lipase and colipase activities in rat intestinal content also increased during the CCK-8 infusion. The enzyme activities reached the maximal level after 30 min of CCK infusion and thereafter progressively decreased to basal levels, remaining there during the following 2 h. The basal level of intestinal enterostatin in rats fed with standard pellets was found to be increased from 1.42 +/- 0.14 to 3.86 +/- 0.4, 3.17 +/- 0.54, and 5.02 +/- 1.6 microM on days 1, 3, and 7, respectively, after high-fat feeding. Parallel to the increase in intestinal enterostatin, there was a significant increase in pancreatic lipase and colipase activities in the intestine during the ingestion period of high-fat diet as compared with the control group. The estimated molecular mass of enterostatin immunoreactivity of intestinal content was similar to that of the synthetic pentapeptide. These results suggest that immunoreactive enterostatin (Val-Pro-Gly-Pro-Arg) is normally present in rat intestinal content, is significantly increased after stimulation with CCK-8, and is also increased after prolonged high-fat feeding.

Amino Acid Sequence↗
PubMed1995

Solution structure of porcine pancreatic procolipase as determined from 1H homonuclear two-dimensional and three-dimensional NMR.

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by 1H NMR using two- and three-dimensional measurements. The secondary structure determination identified two separate three-stranded beta-sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non-trivial proton--proton distance constraints, 14 hydrogen bond distance constraints and 55 phi and 25 X1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N- and C-terminal region. The remaining part of the structure is well defined with a root-mean-square deviation (rmsd) relative to the average of 0.09 +/- 0.02 nm for backbone atoms (residues 11-30, 37-50, 57-69, 83-89). The protein comprises two identical domains, each containing a three-strand beta-sheet and two disulfide bonds: a 15-residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase.

Amino Acid Sequence↗
PubMed1995

Cloning and expression in insect cells of two pancreatic lipases and a procolipase from Myocastor coypus.

The physiological role of pancreatic lipases has traditionally been assigned solely to triacylglyceride metabolism, while the digestion of phospholipids requires the presence of the pancreatic phospholipase A2, a 14-kDa enzyme unrelated to pancreatic lipases. However, in the guinea pig, it was observed that the pancreatic phospholipase A2 was absent and that a guinea pig pancreatic-lipase-related protein 2 (GPL-RP2) was responsible for phospholipase activity, in contrast to the situation observed in other mammalian species. As the guinea pig is a member of the hystricomorph rodents, it was of interest to investigate if other species within this evolutionary suborder display similar characteristics. The coypu (Myocastor coypus) also a member of the hystricomorph rodents, was chosen for further investigations. The cDNAs encoding two pancreatic lipases and a procolipase from the coypu were cloned, expressed and characterized. One lipase, CoPL-RP2, was identified as belonging to the RP2 subfamily, while the second, CoPL, was found to belong to the classical pancreatic lipase subfamily. Enzymic characterization and sequence data suggest a role for coypu colipase as a specific cofactor for CoPL, while this coypu colipase cannot be an important cofactor for CoPL-RP2 in vivo. Also, the new lipase cDNA sequences were used in a phylogentic analysis to reinvestigate the taxonomical position of the hystricomorph rodents (e.g. coypu and guinea pig) with respect to the myomorph rodents (e.g. rat and mouse).

Amino Acid Sequence↗
PubMed1992

Differential inhibition of fat intake in two strains of rat by the peptide enterostatin.

The effect of enterostatin, the amino-terminal pentapeptide of pancreatic procolipase, on food intake was investigated in two strains of rat, the dietary fat-sensitive Osborne-Mendel (OM) rat and the dietary fat-resistant S5B/Pl rat. After an overnight fast, enterostatin inhibited intake of high-fat (HF) but not low-fat (LF) diets in OM rats, but had no effect in S5B/Pl rats. When fed a macronutrient three-choice diet, OM rats selected fat preferentially, whereas S5B/Pl rats selected carbohydrate. Enterostatin specifically inhibited the intake of fat in OM rats but not S5B/Pl rats fed a three-choice macronutrient diet. The activity of pancreatic colipase was increased in S5B/Pl rats on both HF and LF diets compared with OM rats. Pancreatic colipase activities were negatively related to the voluntary intake of fat on three-choice macronutrient diets. The data support the hypothesis that enterostatin may control the intake of dietary fat.

Animals↗