Interactions of colipase with bile salt micelles. 1. Ultracentrifugation studies.

A detailed investigation by ultracentrifugation of the colipase-taurodeoxycholate system showed the formation of well-defined mixed associations with a sedimentation coefficient of about 2.2S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor. Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.     

Small-angle neutron scattering study of the association between porcine pancreatic colipase and taurodeoxycholate micelles

Small-angle neutron scattering studies have shown the association of porcine colipase with bile salts micelles to be a lateral one. The molecular structure parameters of the individual components were determined first. A radius of gyration of 13.9 Å is found for colipase, which implies a non-spherical shape for this molecule. The size of taurodeoxycholate micelles is controlled by the ionic strength of the solution. In 0.15 m-NaCl their volume is comparable to that of colipase; they are elongated with an axial ratio of about 2. At higher ionic strengths the elongation of the micelles increases.In 0.15 m-NaCl the complex is found to be an association of one colipase molecule with a volume of detergent corresponding to that of one free micelle. The contrast variation study of the radius of gyration shows that in the complex the centre of masses of the protein and of the detergent are well-separated: a distance between 29 and 45 Å has been estimated. The value of the radius of gyration of the complex at high contrast, and the agreement between the contrast variation analysis and a straightforward application of the parallel axes theorem indicate that the complex is formed by the juxtaposition of the protein and a preformed micelle, which has approximately the same size and shape as a free micelle. There is only one localized surface contact between the protein and the micelle, which implies that colipase possesses a relatively well-defined binding site.     

Effects of colipase and taurodeoxycholate on the catalytic and physical properties of pancreatic lipase B at an oil water interface.

A monolayer reaction system employing tripropionin and siliconized glass beads was used to study the effects of taurodeoxycholate and colipase on the catalytic activity, interfacial stability, and interfacial affinity of porcine pancreatic lipase B (EC 3.1.1.3) The stability and catalytic activity of lipase at the bead-water interface are governed by the same two ionizable groups with pKa values (in the absence of cofactors) of 5.6 and 9.3. Colipase alone or with bile salt caused only a slight perturbation of these values. At low concentrations, 0 to 0.3mM, taurodeoxycholate increases the stability of lipase by 5-fold. At higher concentrations, 0.3 to 0.8 mM, but still below its critical micelle concentration, taurodeoxycholate prevents the adsorption of lipase to the bead-water interface. This appears to be the major mechanism by which this bile salt inhibits lipolysis. Colipase exerts small positive effects on lipase stability and catalytic activity. More importantly, colipase enables the adsorption of lipase in the presence of bile salt, thereby reversing the inhibition.     

Competitive inhibitory effect exerted by bile salt micelles on the hydrolysis of tributyrin by pancreatic lipase.

Colipase was readily adsorbed on tributyrin particles emulsified with sodium taurodeoxycholate used above its critical micellar concentration. The ensuing rate of lipase adsorption on tributyrin depended on the amount of colipase already adsorbed at the interface. A molar excess of colipase over lipase was required to observe lipolysis at maximal velocity. Bile salt micelles competitively inhibited this reaction.     

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.     

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.     

The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.     

A spectrophotometric assay for the characterization of the S'' subsite specificity of alpha-chymotrypsin

Pig and horse colipases contain three tyrosine residues. In addition, horse colipase possesses a tryptophan residue. Some of the tyrosine residues are involved in the association of colipase and a bile salt micelle. The present report demonstrates that the aromatic residues responsible for colipase fluorescence are in an aqueous environment. In the presence of bile salt micelles, changes in colipase fluorescence properties indicate that the intrinsic fluorophores are located in a more hydrophobic environment upon colipase-micelle complex formation. In addition, the fluorescence of an NBD group fixed on lysine 60, which is very close to the aromatic region in the pig colipase, is also altered in the presence of micelles. These results show that the micelle binding site is not limited to the tyrosine residues but may be broadened to adjacent residues such as lysine 60 and also tryptophan 52 in horse colipase.     

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.     

Neutron crystallographic evidence of lipase-colipase complex activation by a micelle.

The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.     

Effects of gum arabic on lipase interfacial binding and activity.

We investigated the surface behavior of gum Arabic (GA) as well as its effects on the lipolytic activity of human pancreatic lipase (HPL) and Humicola lanuginosa lipase (HLL), using emulsions of triacylglycerols (TAG) with various chain lengths. The effects of GA on the interfacial binding of HPL were also investigated. In the presence of 4 mM sodium taurodeoxycholate (NaTDC), GA (3% w/v, final concentration) had no effect on the HPL activity measured in the presence of colipase, whatever the type of TAG used. However, in the absence of bile salts or at low bile salt concentrations, GA inhibited the HPL activity when trioctanoin (TC8) and purified soybean oil (PSO) were used as substrates. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. Both crude and dialyzed GA solutions were found to be highly tensioactive at the air-water as well as the oil-water interface using the drop technique. In conclusion, GA, or a putative contaminant present in GA, was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL.     

The interaction of bile salt micelles with the dansyltyrosine derivatives of porcine colipase

The interaction of bile salt micelles with the tyrosines of pancreatic colipase was assessed by steady-state and time-resolved fluorescence techniques. Dansyltyrosine fluorescence showed that Tyr-55 was located in the proposed interface recognition site. In support of this claim was a 70 nm blue shift and 4.3-fold quantum yield increase in emission spectrum due to taurodeoxycholate (TDOC) micelle-complex formation. Complex formation also caused a shift in the center of the major lifetime distribution from 11.7 to 15.1 ns, and more than doubled the polarization and anisotropy decay parameters. These data supported an earlier model of colipase-micelle binding that suggested that Tyr-55 was inserted into the interior of the TDOC micelle upon binding (J.C. McIntyre, P. Hundley and W.D. Behnke, Biochem. J. 245 (1987) 821). Identical experiments on a DNS-Tyr-59 derivative of colipase showed that Tyr-59 did not specifically interact with micelles. Moreover, acrylamide quenching data suggest an alteration in the protein environment surrounding DNS-Tyr-59 such that during complex formation, the efficiency of quenching of DNS-Tyr-59 increases.     

Human pancreatic lipase: colipase dependence and interfacial binding of lid domain mutants

Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.     

A multitechnique approach in protein/surfactant interaction study: physicochemical aspects of sodium dodecyl sulfate in the presence of trypsin in aqueous medium

Interaction of sodium dodecyl sulfate (SDS) with a globular protein, trypsin, has been physicochemically studied in aqueous medium in detail using tensiometric, conductometric, calorimetric, fluorimetric, viscometric, and circular dichroism techniques. The results indicate that SDS-trypsin aggregates start to form at a surfactant concentration higher than the critical micelle concentration of pure SDS micelle. In contrast, the counterion binding decreases in the presence of trypsin. The free energies and enthalpies of micellization, interfacial adsorption, and entropy of micellization associated with the interaction have also been calculated. The values show that the interaction phenomenon is entropy controlled and endothermic in nature. The increase in viscosity is observed for the system of SDS-trypsin cluster above the critical micelle concentration of SDS micelle only. The aggregation number and interface polarity decrease compared to the values of micelles without protein. Circular dichroism spectra show the high alpha-helical content and unfolded structure of trypsin in the presence of SDS due to strong electrostatic repulsion leading to a probable "necklace and bead" model in the case of biopolymer-surfactant complexes.     

Interactions between bovine myelin basic protein and zwitterionic lysophospholipids.

The binding of myelin basic protein to lysolauroylphosphatidylcholine (lysoLPC) and lysolauroylphosphatidylethanolamine was investigated at neutral pH using gel partition chromatography and equilibrium dialysis at 20 and 37 degrees C. The results show that the protein-lysolipid interactions are highly cooperative and that the free lysolipid concentration at which the binding commences is markedly influenced by both the chemical structure of the lysolipids and the temperature. The binding begins just below the critical micelle concentration for both lysolipids, which suggests that the forces governing micellization and the binding are similar. Circular dichroism (CD) spectroscopy was used to follow changes in the conformation of the protein caused by lysomyristoylphosphatidylcholine and lysoLPC. The CD results indicate that lysolipid association with the protein commences below the critical micelle concentration and continues above this concentration. Mechanisms for the lysolipid-protein interaction, which are consistent with the binding and CD data, are discussed.     

Magnetic-circular-dichroism studies of haem a and its derivatives.

1. The Thy-1 membrane glycoproteins from rat thymus and brain bound deoxycholate to 24% of their own weight as measured by equilibrium dialysis. The binding occurred co-operatively at the critical micelle concentration of deoxycholate, suggesting that the glycoproteins bind to a micelle, and not to the detergent monomer. 2. From sedimentation-equilibrium and deoxycholate-binding data the molecular weights of the glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoproteins respectively. The molecular weight of the polypeptide part of the glycoprotein is thus 12500. 3. In the absence of deoxycholate, brain or thymus Thy-1 glycoprotein formed large homogeneous complexes of mol. wt. 270000 or 300000 respectively. The sedimentation coefficient of these was 12.8 S. The complex was only partially dissociated by 4M-guanidinium chloride. 4. After cleavage of brain or thymus Thy-1 glycoprotein with CNBr, two peptides were clearly identified. They were linked by disulphide bonds and both contained carbohydrate. This cleavage suggests there is only one methionine residue per molecule, which is consistent with the above molecular weights and the known amino acid composition.     

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase.

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.     

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.     

Differential refractometric determination of binding of sodium dodecyl sulfate to protein using high-performance gel chromatography

When sodium dodecyl sulfate (SDS) is added to a high-performance gel chromatographic column equilibrated with a buffer solution containing SDS at a level above the critical micelle concentration, the surplus SDS migrates as micelles giving a sharp peak. The presence of an unfolded protein in the sample solution gives a polypeptide peak in advance of the SDS micelle peak. As the result of SDS binding to the polypeptide, the SDS micelle peak is attenuated in comparison to that in the absence of protein. Thus the amount of SDS bound to the polypeptide can be determined accurately and simply from the decrease in the area of the SDS micelle peak. This approach is particularly useful for precise determination of bound SDS, which is pertinent to understanding the state of the protein polypeptide-SDS complex under the conditions of SDS-polyacrylamide gel electrophoresis.     

Interactions of free and immobilized myelin basic protein with anionic detergents

The interaction of free and immobilized myelin basic protein (MBP) with sodium deoxycholate (DOC) and sodium dodecyl sulfate (NaDodSO4) was studied under a variety of conditions. Free MBP formed insoluble complexes with both detergents. Analysis of the insoluble complexes revealed that the molar ratio of detergent/MBP in the precipitate increased in a systematic fashion with increasing detergent concentration until the complex became soluble. At pH 4.8, equilibrium dialysis studies indicated that approximately 15 mol of NaDodSO4 could bind to the protein without precipitation occurring. Regardless of the surfactant, however, minimum protein solubility occurred when the net charge on the protein-detergent complex was between +18 and -9. Complete equilibrium binding isotherms of both detergents to the protein were obtained by using MBP immobilized on agarose. The bulk of the binding of both detergents was highly cooperative and occurred at or above the critical micelle concentration. At I = 0.1, saturation levels of 2.09 +/- 0.15 g of NaDodSO4/g of protein and 1.03 /+- 0.40 g of DOC/g of protein were obtained. Below pH 7.0 the NaDodSO4 binding isotherms revealed an additional cooperative transition corresponding to the binding of 15-20 mol of NaDodSO4/mol of protein. Affinity chromatography studies indicated that, in the presence of NaDodSO4 (but not in its absence), [125I]MBP interacted with agarose-immobilized histone, lysozyme, and MBP but did not interact with ovalbumin-agarose. These data support a model in which the detergent cross-links and causes precipitation of MBP-anionic detergent complexes. Cross-linking may occur through hydrophobic interaction between detergents electrostatically bound to different MBP molecules.