Colipase Adsorption Research Articles

Pectin emulsions for colon-targeted release of propionic acid

In this study, we aimed to design emulsion system, using pectin as an emulsifier that could resist gastric and intestinal digestion, and deliver tripropionin (glyceryl tripropionate) as propionic acid precursor to the colon. The stability and the degree of lipolysis of the emulsions under in vitro gastrointestinal digestion were assessed using droplet sizing, microscopy, ζ-potential and quantification of tripropionin, propionic acid and pectin coverage. The emulsions were stable in the gastric stage, and the stability under intestinal conditions was dependent on the concentration of pectin. The microscopic structure demonstrated a certain degree of coalescence of all emulsions after intestinal digestion, with the most affected emulsion being that at the lowest pectin concentration. Pectin concentrations of ≥2.5 wt% improved the resistance of the emulsion to coalescence and reduced the rate of lipolysis and the degree of hydrolysis, all of which were due to thicker pectin coverage. This apparently improved the resistance to displacement by bile salts and the subsequent adsorption of colipase and lipase that occur in the initial phase of lipid digestion. The use of pectin to form gastrointestinal-resistant emulsions is a promising approach to delaying the digestion of tripropionin, or other lipid-based materials, and to deliver tripropionin to the colon.

Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile–salt–oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I330/I350 ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.

Model of Peripheral Protein Adsorption to the Water/Lipid Interface

Two models have been developed to describe the adsorption of a model peripheral protein, colipase, to phospholipid/diacylglycerol (PL/DG) monolayers. One model is applicable at monolayer collapse pressure and at any composition that exceeds the DG mole fraction of PL/DG lateral complexes (Sugár, I. P.; Mizuno, N. K.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2001, 81, 3387-3397). The other model is applicable at any lateral pressure but only below the mole fraction of DG in the complex (Sugár, I. P.; Mizuno, N. K.; Brockman, H. L. Biophys. J. 2005, 89, 3997-4005). Both models assume that initiation of colipase adsorption to the water/lipid interface requires an area of water-exposed hydrophobic surface that exceeds a critical value. In the first model, accessible surface is provided by the head groups of the uncomplexed DG molecules. This surface area follows a binomial distribution. In the second model, accessible area is created by hydrocarbon chains becoming exposed at the water/lipid interface as total lipid packing density of monolayers of PL and/or PL/DG complexes is decreased. This surface area follows a Poisson distribution. The model described in this paper is a unification, extension, and improvement of these models that is applicable at any lateral pressure and any PL/DG mole fraction. Calculated normalized initial colipase adsorption rates are compared with the available experimental values, and predictions of the adsorption rates are made for currently unmeasured compositions and lateral pressure regimes.

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.

Regulation of lipases by lipid–lipid interactions: implications for lipid-mediated signaling in cells

Lipases are extracellular peripheral proteins that act at the surface of lipid emulsions stabilized, typically, by phospholipids. At a critical composition lipase activity toward substrates in phospholipid monolayers is discontinuously switched on by a small increase in substrate mole fraction. This occurs in part because lipase binding is inhibited by phospholipids. Binding of the lipase cofactor, colipase, is also inhibited by phospholipids. The initial rate of colipase binding increases abruptly at a substrate mole fraction that is approximately half the critical composition for lipase activity and just above that in substrate-phospholipid complexes. Moreover, complex collapse areas show an ∼1:1 correlation with phospholipid excluded areas determined from an analysis of colipase adsorption rates. Thus, complexes inhibit colipase binding rate. Additionally, the switching of lipase activity likely occurs when uncomplexed substrate becomes the majority species in the interface. Lipase substrates, e.g. diacylglycerols, are typically the same lipids generated in the cytoplasmic surface of the plasma membrane of stimulated cells. As colipase binding is nonspecific and complexes involving lipase substrates form on the basis of lipid–lipid interactions alone, complexes should form in the plasma membrane of stimulated cells and may regulate protein translocation to the membrane.

Phospholipid−Diacylglycerol Complexes Regulate Colipase Adsorption to Monolayers

Monomolecular films of a 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and any of several non-phospholipids, like diacylglycerols and free fatty acids, inhibit the initial rate of adsorption of a...

Colipase-induced reorganization of interfaces as a regulator of lipolysis

Colipase is a 10 kDa protein cofactor that allows pancreatic triacylglycerol lipase (PTL) to function in the presence of endogenous inhibitors, like diacyl phosphatidylcholine. From structural studies of lipase–colipase it has been shown that colipase forms a 1:1 complex with lipase, increases the hydrophobic surface area of lipase presented to the interface and exposes the active site. This has led to the idea that colipase serves to anchor lipase at inhibitor-rich interfaces, thereby allowing lipase to function catalytically. Early studies, however, present a more complicated picture of lipolysis and indicate a role for the products of lipolysis in the anchoring and activation of lipase. Using techniques of surface chemistry, we have studied the adsorption of pancreatic lipase and colipase to defined lipid monolayers comprised of SOPC and model lipolysis reactants, i.e . fatty acid and 1,2-diacylglycerol. At physiologically relevant lipid packing densities, lipase does not adsorb to phosphatidylcholine unless fatty acid is also present. Moreover, in phosphatidylcholine-rich interfaces containing fatty acid, as might be encountered in vivo, lipase adsorption is greatly enhanced by the presence of colipase. To determine why this occurs, colipase adsorption to monolayers of phosphatidylcholine and lipolysis reactants were compared. We found that colipase interacts more strongly with interfaces comprised of or containing lipolysis reactants than those of phosphatidylcholine. Analysis of the interaction stoichiometry indicates that ∼25 reactant acyl chains are affected by the presence of colipase in the interface and suggests that colipase laterally reorganizes the interface, concentrating lipolysis reactants in its vicinity. To test this hypothesis directly, the fluorescence properties of BODIPY-containing lipids in monolayers were characterized in the presence and absence of colipase. Preliminary results support the reorganization hypothesis. Overall, the studies reviewed herein support the idea that colipase laterally concentrates reactants of lipolysis in its vicinity, creating a reactant-rich nanodomain to which lipase adsorbs.

Lipid Lateral Organization in Fluid Interfaces Controls the Rate of Colipase Association

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.

Lateral packing of the pancreatic lipase cofactor, colipase, with phosphatidylcholine and substrates.

The interaction of the pancreatic lipase cofactor colipase with a diacylphosphatidylcholine, acylglycerols, and free fatty acid was investigated by monitoring its adsorption to monomolecular lipid films. Surface pressure and colipase surface concentration were measured as a function of the initial lipid concentration and composition. Colipase adsorbs to a level of 28-30 pmol/cm2 to form a close-packed monolayer of protein and interacts strongly with all lipids when the lipid chain:colipase ratio is </=3. Consideration of the size difference between the protein and acyl groups suggests that in this regime the lipid is occupying the voids between tightly packed protein molecules. At lipid chain:colipase ratios >3, the triacylglycerol is excluded from the monolayer phase. Phosphatidylcholine, diacylglycerols, and free fatty acid remain in the monolayer phase up to </=25 lipid chain:colipase ratios. Geometrically over this range of compositions, the colipase molecules should be separated by up to 0-2 acyl chains. At higher lipid chain:colipase ratios, diacylglycerols are likely excluded from the monolayer phase. Anomalous behavior is observed with the fatty acid which at lipid chain:colipase ratios >25 induces higher levels of colipase adsorption than at lower ratios. This suggests the formation of a novel structure involving fatty acid and/or colipase. Phosphatidylcholine also remains in the interface at lipid chain:colipase ratios >3 but shows little additional interaction with colipase. However, fluorescence microscopy suggests that the phosphatidylcholine and colipase are miscible in the interface. The specificity demonstrated in this study suggests that colipase may regulate the type of surfaces to which colipase and, hence, lipase bind and may control the species distribution of substrate to which bound lipase is exposed.