Adsorption Of Lipase Research Articles

Characterization of the serine reacting with diethyl p-nitrophenyl phosphate in porcine pancreatic lipase

The position in porcine pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) of the serine reacting specifically with emulsified or micellar diethyl p-nitrophenyl phosphate has been investigated. This serine which appears to be involved in lipase adsorption to insoluble triglyceride interfaces, is at position 152 in the enzyme chain. The sequence around this amino acid is: His-Val-Ile-Gly-His-Ser-Leu-Gly.

Effect of taurodeoxycholate, colipase and temperature on the interfacial inactivation of porcine pancreatic lipase.

Beside their inhibitory effect upon lipase adsorption, bile salts at low concentration (around 0.2 mM) have repeatedly been shown to enhance lipolysis slightly. From the data reported in this paper, this activation has been attributed to a stabilization of the adsorbed lipase brought about by low concentrations of bile salts. This hypothesis relies on the following observations. (a) For a given temperature, the activation by bile salts depends on the substrate. It is maximum for trihexanoin (trihexanoylglycerol) and does not exist for tripropionin (tripropionylglycerol). (b) In the absence of bile salts, the optimal activities are obtained for different temperatures depending on the substrate. (c) For a given substrate, the activation by a low concentration of bile salts depends on the temperature. It increases when the temperature is raised (up to 35-40 degrees C) and completely disappears at a sufficiently low temperature (around 10 degrees C). (d) This temperature effect does not seem to be due to a modification of the physical parameters of the interface as measured by the interfacial tension. (r) Colipase, like bile salts, increases the lipase activity on short-chain triglycerides but only at high temperature when lipase denaturation occurs. It has no influence upon the activity when the temperature is sufficiently low (around 10 degrees C).

Effects of bile lipids on the adsorption and activity of pancreatic lipase on triacylglycerol emulsions

Emulsions of natural triacylglycerols obtained with different shear forces were used to study lipase adsorption and lipolysis. The influence of the bile lipoprotein complex on these two processes was determined. Optimal lipase activity was observed to occur with a given phospholipid: triacylglycerol ratio. This ratio depended on the degree of triacylglycerol emulsification and was accompanied by maximal adsorption of the bile lipoprotein complex. These results support our previous model for pancreatic lipolysis under physiological conditions, according to which colipase controls lipase binding to the bile lipoprotein complex and the resulting association directs enzyme adsorption to the acylglycerol particle (Lairon, D., Nalbone, G., Lafont, H., Léonardi, J., Domingo, N., Hauton, J.C. and Verger, R. (1978) Biochemistry 17, 5263–5269).

Enzyme-substrate interaction in lipid monolayers. I. Experimental conditions and fundamental kinetics.

A systematic study has shown the importance of the different factors which are concerned with the action of lipase on a substrate (1,3-didecanoylglycerol). These consist of a) the process of adsorption of lipase to the surface, b) the necessity of limited stirring to reach equilibrium, and c) the persistence during the reaction process of the enzyme molecules adsorbed on the monolayer. On the basis of this preliminary investigation, a technique was established to analyze the mechanism of lipase action with defined quantities of enzyme and lipid segregated in the monolayer. Thus, the process of the reaction itself is separated from the adsorption process, and it is demonstrated that the quantity of substrate hydrolyzed per minute depends only on the quantity of initially adsorbed lipase and not on the quantity of substrate or on the surface concentration of the enzyme. An appropriate new definition of the rate is consequently adopted.

Possible roles of bile lipids and colipase in lipase adsorption.

The adsorption isotherms of bile salts, phospholipids, and cholesterol were determined with siliconized glass beads. It was observed that the molar fractions of cholesterol, phospholipid, and bile polypeptide fractions increased simultaneously and considerably on the surface of the beads in comparison to the corresponding fractions found in bile. The composition of the adsorbed film is approximately 1 cholesterol: 2 phospholipid: 3 bile salt molecules. The performed complex of lipase, colipase, and bile lipids behaves as an entity which determines lipase adsorption. The modification of the interface quality of a lipid substrate by a detergent is not perse the reason for the lack of lipase adsorption. A model is proposed according to which lipolysis under physiological conditions would occur in two steps requiring two cofactors. Colipase would be necessary for the formation of the lipase-bile lipoprotein complex, and bile lipids would be required to direct the adsorption of this lipolytic entity toward the emulsified substrate.

Inhibition of lipase adsorption at interfaces. Role of bile salt micelles and colipase.

The effects of bile salts and colipase on the adsorption of lipase at an interface were studied by hydrophobic affinity chromatography on phenyl- and octyl-Sepharose. In the absence of bile salts, lipase or colipase binds separately to the gel. This is unchanged in the presence of adsorbed bile salts, when one bile salt molecule is associated per hydrophobic ligand. The same data are obtained in the presence of monomeric bile salt solutions. In contrast, lipase adsorption is totally prevented in a micellar bile salt solution. These results favor the idea that the formation of a lipase-bile salt complex in solution is responsible for the lack of interfacial lipase adsorption.

Inhibition of lipase adsorption at interfaces. Role of bile salts micelles and colipase.

Inhibition of lipase adsorption at interfaces. Role of bile salts micelles and colipase.

Affinity chromatography of lipase with hydrophobic ligands coupled to cyanogen bromide-activated agarose

The behavior of lipase produced by Pseudomans mephitica var. lipopytica toward hydrophobic residues coupled to spacer gels that were prepared by coupling a primary amine to CNBr-activated agarose, was studied. The lipase adsorbed on the ligand of a long unbranched aliphatic chain, a benzene ring, or deoxycholic acid was only slightly or not all eluted at pH 5 or pH 11 by buffers containing 1 M NcC1. The lipase was eluted by liquid containing a surfactant or an organic solvent miscible with water, indicating greater involvement of hydrophobic forces. The adsorption of propane, cyclopentane, cyclohexane, cycloheptane, or chrysene appears to be achieved through electrostatic forces, inasmuch as desorption was caused by buffer containing 1 M NaC1 at pH 11. The amount of lipase adsorbed on these hydrophobic ligands was about the same as that adsorbed on the ligands belonging to the first group. Since little lipase wad adsorbed on cyclopropane, cycloctane, pyridine, methane, n-pentane, or branched aliphatic chains, these ligands appear to impose steric hindrance on the adsorption of lipase, or they may be too small to fit into the hydrophobic sites of lipase.

Competitive inhibitory effect exerted by bile salt micelles on the mydrolysis 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.

Studies on the mechanism of lipase reaction. III. Adsorption of Chromobacterium lipase on hydrophobic glass beads.

The lipase from Chromobacterium was adsorbed on glass beads which was coated with olive oil, liquid paraffin or silicone oil. These adsorption was treated in the Lineweaver-Burk's plot and characters of the adsorption were similar each other regardless of their chemical structure of hydrophobic materials. On the other hand, esterase from porcine liver was not adsorbed on hydrophobic glass beads. The interaction between the lipase and hydrophobic surface conformed to the Langmuir's adsorption isotherm with a dissociation constant K=1.4×10-7M. At saturation of the surface with the lipase each protein molecule occupies an average area of 4500 A2 per molecule. The lipase adsorbed on hydrophobic surface did not inactivated but activated about 3-fold. It was elucidated that the hydrophobic bond play a major role in the adsorption of the lipase on substrate or hydrophobic surface.

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.

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.

Role of colipase in the interfacial adsorption of pancreatic lipase at hydrophilic interfaces

Role of colipase in the interfacial adsorption of pancreatic lipase at hydrophilic interfaces

Studies on the mechanism of the lipase reaction: II. Comparative studies on the adsorption of lipases and various proteins at the air-water interface

Adsorption of lipases (EC 3.1.1.3) and various proteins at the air-water interface has been investigated in relation to the mechanism of lipase reaction. Aqueous solutions of lipases and denaturated proteins show surface activity as strong as that of synthetic detergents. However, the surface activity of esterases and various other proteins is little or none. By foam fractionation it was shown that lipases were adsorbed at the air-water interface and the adsorption followed the equation of Langmuir's adsorption isotherm. The properties of lipase at the interface are discussed in relation to the mechanism of lipase reaction and the differences from the esterase reaction.

Effect of colipase on adsorption and activity of rat pancreatic lipase on emulsified tributyrin in the presence of bile salt

The present work was undertaken to examine rat pancreatic lipase activity in relation to its ability to be adsorbed on emulsified tributyrin. This study was conducted at a supramicellar concentration of sodium taurodeoxycholate, between pH 6.0 and 8.0, and at different colipase concentrations. A pH adsorption curve was differentiated from the pH activity curve of lipase. The authors concluded that the socalled acid shift of the optimal pH for lipase action described earlier is due to the low adsorption rate of lipase on its substrate at alkaline pH rather than to a change of the pH dependence of the V(max) and K(m) of the enzyme.

Sur le role des ions calcium durant l'hydrolyse des triglycerides insolubles par la lipase pancreatique en presence de sels biliares

Resume Porcine pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3) is not active on long chain triglyceride emulsions stabilized by deoxycholate unless calcium ions are present. The amount of lipase bound at the interface of the emulsified particles can be determined by measuring the activity remaining in the aqueous phase after centrifugation of the emulsion. It was shown in this way that, when bile salts are present, calcium is necessary for the adsorption of lipase at the triglyceride-water interface in the alkaline pH range where the enzyme is normally active. A determination of the two parameters Km and V of the reaction indicates that Km varies proportionally to the reciprocal value of the Ca2+ concentration whereas V remains constant. Hence, calcium is bound by lipase rather than by the substrate. The Km of the reaction pH 9.0 and 37° is 0.25 ± 0.3 m2/l in the presence of 0.3 mM Ca2+. Under the same conditions, the association constant of calcium for lipase is 1.2 · 104 (moles/l)−1 and the association constant of the complex lipase-calcium for the emulsified substrate is 5.0 (m2/l)−1. It is possible that the role of calcium is to compensate the electrostatic repulsion existing between the negatively charged lipase molecule and the ionised carboxyls of the bile salts situated at the interface of the emulsified triglyceride particles.

Etude cinetique de l’action de la lipase pancreatique sur des triglycerides en emulsion. Essai d’une enzymologie en milieu heterogene

Summary The purpose of this paper was to study the action of pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3) on emulsified triglycerides. This type of action is an interesting example of an enzymatic process taking place in an heterogeneous medium. It is shown that: 1. Lipase is adsorbed by its emulsified substrate and the initial rate ( v ) of the reaction is a function of the number of enzyme molecules adsorbed at the interface. 2. By varying the dilution of a triglyceride emulsion in bile salts in the presence of 0.1 M NaCl, linear Lineweaver-Burk representations are obtained from which the reaction paramaters V m arid K m can easily be derived. However, the abscissae of the representation, and consequently the K m values, remain undefined as long as a right substitute for the substrate concentration concept is not found. Obviously, this concept cannot be used when the substrate is in an insoluble state. 3. By using two emulsions containing particles of different size, it can be shown that v does not depend directly on the weight of the insoluble substrate. For the same weight, the same amount of lipase and the same experimental conditions. v becomes higher when the average size of the particles decreases, v depends on the area ( I ) of the interface. I can be experimentally determined with a good accuracy by two techniques : centrifugation of the emulsions in a sucrose gradient and counting of the particles according to their size in an electronic Coulter counter. 4. On the other hand, v is inversely proportional to the total volume ( A 0 ) of the emulsion. Thus, lipase adsorption may be considered as fully reversible in our experimental conditions and to take place according to Langmuir's isotherms. The “interface concentration” I / A 0 , i.e. the interfacial area in a volume unit of emulsion, is playing, in the case of lipase, the role normally devoted to the concentration of the substrate. The K m (emulsion) of lipase corresponds to a certain value of this interface concentration. This does not mean that I / A 0 is the only significant variable in the process. Other variables such as electrical charge of emulsified particles and molecular organization at the interface may also play a role. 5. It may be supposed that the well-known influence of bile salts during the intraluminar phase of triglyceride digestion is at least partly related to the ability of these substances to favour the reversibility of lipase adsorption at the interface.