Binding of bile salts to pancreatic colipase and lipase.

The binding of conjugated bile salts to pancreatic colipase and lipase has been studied by equilibrium dialysis and gel filtration. The results indicate that at physiological ionic strength and pH, conjugated bile salts bind as micelles to colipase: 12-15 moles/mole of colipase for the dihydroxy conjugates and 2-4 for the trihydroxy conjugates. No binding of bile salt takes place from monomeric solutions. Under the same experimental conditions, only 1-2 moles of conjugated dihydroxy bile salts bind to pancreatic lipase.     

Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase

Efficient dietary fat digestion is essential for newborns who consume more dietary fat per body weight than at any other time of life. In many mammalian newborns, pancreatic lipase related protein 2 (PLRP2) is the predominant duodenal lipase. Pigs may be an exception since PLRP2 expression has been documented in the intestine but not in the pancreas. Because of the differences in tissue-specific expression, we hypothesized that the kinetic properties of porcine PLRP2 would differ from those of other mammals. To characterize its properties, recombinant porcine PLRP2 was expressed in HEK293T cells and purified to homogeneity. Porcine PLRP2 had activity against tributyrin, trioctanoin and triolein. The activity was not inhibited by bile salts and colipase, which is required for the activity of pancreatic triglyceride lipase (PTL), minimally stimulated PLRP2 activity. Similar to PLRP2 from other species, PLRP2 from pigs had activity against galactolipids and phospholipids. Importantly, porcine PLRP2 hydrolyzed a variety of dietary substrates including pasteurized human mother's milk and infant formula and its activity was comparable to that of PTL. In conclusion, porcine PLRP2 has broad substrate specificity and has high triglyceride lipase activity even in the absence of colipase. The data suggest that porcine PLRP2 would be a suitable lipase for inclusion in recombinant preparations for pancreatic enzyme replacement therapy.     

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

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

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.     

The effect of pancreatic procolipase and colipase on pancreatic lipase activation

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.     

Separation and characterization of the precursor and activated forms of porcine and human pancreatic colipase by reversed-phase liquid chromatography

Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.     

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.     

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.     

Inhibition of pancreatic lipase B activity by taurodeoxycholate and its reversal by colipase.

In our two-phase reaction system taurodexycholate prevents the adsorption of pancreatic lipase B to the nonaqueous phase. Our data are consistent with a mechanism for this reaction which involves the cooperative formation of an enzyme-(bile salt)4 complex in solution with a dissociation constant of 1.4 X 10(-15)M4. Whereas the free enzyme is readily adsorbed to a bile salt-substrate-covered surface, the complex is not. Thus, the "inhibition" of substrate hydrolysis occurs because enzyme and substrate are separated physically. The protein cofactor, colipase, reverses the inhibitory effects of bile salt by providing a high affinity binding site at the interface for the lipase-(bile salt)4 complex. Steady state and presteady state kinetic data are consistent with the formation of a complex with a 1/1, lipase/colipase, ratio, and a dissociation constant of 0.4 to 2.8 X 10(-9)M. The rate of adsorption of lipase to adsorbed colipase appears to be controlled by diffusion through the unstirred layer with a second order rate constant of 1.3 X 10(6)M-1S-1.     

Synthesis and characterization of the dansyltyrosine derivatives of porcine pancreatic colipase

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

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.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

Construction and expression of synthetic wild-type and mutant genes encoding porcine pancreatic colipase: tryptophan fluorescence studies

Based on the known (95-residue) amino acid (aa) sequence of porcine pancreatic colipase (CLP), a cofactor of pancreatic lipase, a 297 bp gene was designed and assembled from eight synthetic, overlapping DNA fragments. Optimized for expression in bacteria, the CLP-encoding gene (CLP) was inserted into the lacZ gene fragment contained in the small expression vector, pUC8, and cloned in Escherichia coli JM109. Expression of this construct yielded a protein approx. 11 kDa in size, equivalent to CLP, with an Mr of 10,336, plus ten additional amino acids at the N-terminus. The recombinant CLP (reCLP) was solubilized from bacterial inclusion bodies and then purified and refolded. A mutant CLP gene, changing Tyr-55 to Trp, was then constructed by site-directed mutagenesis. Since porcine CLP contains no Trp, this strategy provided a protein with an internal fluorescent probe for biophysical studies. The presence of Trp in the mutant protein was confirmed using fluorescence spectroscopy. Both wild-type (wt) and mutant reCLP reacted on Western blots with an affinity-purified rabbit anti-CLP antibody, raised against native CLP. The Tyr-55 to Trp exchange did not affect the activity of reCLP. Fluorescence studies of the interaction between reCLP and the bile salt, taurodeoxycholate (TDOC), showed that Trp-55 in the hydrophobic binding site of mutant reCLP inserted into the interior of the bile salt micelle.     

Purification of porcine enterokinase by affinity chromatography.

A method is described for the purification of porcine enterokinase by affinity chromatography with p-aminobenzamidine as the ligand. Purification was completed by immunoadsorption with antisera raised to components binding non-biologically to the gel. The final enterokinase preparation was 2.3 times more active than the most active preparation previously described.     

Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems

The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.