Regulation of carboxylester lipase adsorption to surfaces. 1. Chemical specificity

The chemical specificity of the adsorption of porcine pancreatic carboxyl ester lipase to pure lipid surfaces was examined. Adsorption of native and catalytically inactivated enzyme was measured at the argon-buffer interface by using lipid films near the point of collapse. Protein adsorbed readily to films of triolein, 1,3-diolein, methyl oleate, oleonitrile, oleyl alcohol, and 13,16-docosadienoic acid. However, recovery of enzyme activity was variable. These differences and the changes in surface pressure accompanying adsorption indicated the occurrence of enzyme denaturation at the interface. Denaturation was controlled largely by surface free energy but showed some chemical specificity at high surface pressures. Adsorption of protein to the lipids was comparable when measured under either equilibrium or initial rate conditions. Together with surface pressure changes that accompany adsorption, the data indicate a relative lack of specificity for the enzyme-surface interaction. Adsorption to 13,16-docosadienoic acid and 1,3-diolein obeyed the Langmuir adsorption isotherm. Dissociation constants ranged from 10 to 50 nM, depending on enzyme form, ionic strength, and pH. With both lipids, a monolayer of enzyme was adsorbed at saturation. In contrast to these results, adsorption of enzyme activity and protein to films of 1-palmitoyl-2-oleoyl-phosphatidylcholine was less than or equal to 5% of that observed with the other lipids under all conditions. Comparison of rate constants for adsorption to 13,16-docosadienoic and 1,3-diolein as a function of subphase pH indicated a marked dependence on the ionization state of the fatty acid. Overall, the data suggest that the presence of zwitterionic and anionic lipids may regulate the interaction of the enzyme with substrate-containing surfaces in vivo.     

Lipid-lipid interactions as regulators of carboxylester lipase activity

The hydrolysis of 1,3-dioleoylglycerol and related substrates by mammalian pancreatic carboxylester lipases was studied. Mixed lipid films of substrates with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at the argon-buffer interface were exposed to relatively high levels of monomeric porcine pancreatic carboxylester lipase for a brief period. With either 1,3-dioleoylglycerol, 1,2-dioleoylglycerol, trioleoylglycerol, or oleoylmethanol as a substrate, the percentage of substrate hydrolysis increased abruptly from near zero to near 100% with increasing proportion of substrate in the film. The phospholipid was not hydrolyzed. Using 1,3-dioleoylglycerol as the substrate with either the dimeric, porcine pancreatic carboxylester lipase, human pancreatic carboxylester lipase, or human milk bile salt-stimulated lipase gave results identical to those obtained with the porcine monomer. Hydrolysis of 1,3-dioleoylglycerol by porcine monomeric carboxylester lipase was independent of the initial surface pressure of the film. However, a strong correlation was observed between hydrolysis and interfacial lipid composition at all surface pressures, even if bulk 1,3-dioleoylglycerol was also present. The ultrasensitive dependence of hydrolysis on interfacial lipid composition, i.e. lipid-lipid interactions, suggests that such "switching" may contribute to the regulation of diacylglycerol levels in cells where they function in signal transduction.     

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.     

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 2. Effects of lateral lipid distribution in mixtures with phosphatidylcholine.

The lipase-catalyzed exchange of the carboxyl oxygens of 13,16-cis,cis-docosadienoic acid (DA) was studied in the presence of a nonsubstrate matrix lipid, 1-palmitoyl-2-oleoylphosphatidylcholine. For mixed lipid films at the argon-water interface exposed to pancreatic carboxylester lipase (EC 1.1.1.13), the extent of oxygen exchange showed an abrupt increase as the abundance of DA in the interface was increased from 0.5 to 0.6 mole fraction. This compositional range was independent of the level of enzyme used and of the surface pressure, i.e., lipid packing density, of the film. Concomitant with the transition was a change in the apparent mechanism of exchange from coupled to random sequential. Like the extent of oxygen exchanged, the shift in mechanism was independent of all variables except the lipid composition of the interface. The absence of any chemical or physical change accompanying the exchange reaction precludes mechanistic explanations based on the generation of reaction products by the enzyme. Instead, the results suggest that the lateral distribution of DA in phosphatidylcholine-DA interfaces regulates the expression of carboxylester lipase activity and its apparent mechanism. Preliminary measurements give an average cluster size of 1825 molecules of DA when its mole fraction is 0.35. As the DA content of the interface reaches 0.5-0.6, there appears to be a lipid head-group based percolative transition in which DA becomes the continuum. Because this transition involves the lateral organization of the lipids themselves, other interfacially active enzymes may be regulated similarly.     

Protein adsorption to solid surfaces.

The phenomenon of protein adsorption to solid surfaces affects the performance of many materials and processes, in areas ranging from medicine to biochemical engineering. Controlling protein adsorption, from solutions of single proteins as well as from more complex mixtures, requires an understanding of the mechanism(s) by which it occurs. This, in turn, entails detailed characterization of both the protein and the solid surface and identification of those factors controlling the adsorption process.     

Effect of bile salts on the interfacial inactivation of pancreatic carboxylester lipase

Pig pancreatic carboxylester lipase (cholesterol esterase, E.C. 3.1.1.13) was inactivated at a tributyrin/water interface. The apparent rate constant for inactivation increased with increase in the particle surface area of the tributyrin emulsion. The large energy of activation and entropy change for inactivation (33.7 Kcal.mol-1 and 35.8 cal.mol-1.deg-1, using 5 mM sonicated tributyrin at 37 degrees C, respectively) suggest that the observed inactivation reflects denaturation of the enzyme at the tributyrin/water interface. Bile salts protected the enzyme from irreversible inactivation at the tributyrin/water interface. The protection by bile salts was related both to their concentration and to the tributyrin concentration (substrate surface area). The protection by bile salts was not related to their concentration below or above their critical micellar concentration; the binding of bile salts to enzyme was probably the dominant protection factor. Similar stabilization was observed with other detergents such as Brij-35, Triton X-100, and sodium dodecyl sulfate. These results suggest that inactivation of carboxylester lipase occurs at a high-energy lipid-water interface and that an important role of bile salts in vivo is to stabilize carboxylester lipase at interfaces.     

Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas

Carboxylester lipase (cholesterol esterase, EC 3.1.1.13) has been purified to homogeneity from porcine pancreas. The enzyme is isolated in two molecular mass forms, a monomer of 74 kDa and a dimer of 167 kDa. The dimer consists of two catalytically-active subunits which have molecular masses approximately 9 kDa greater than the monomers. The difference in size was not attributable to carbohydrate or lipid content. The catalytic properties of the two forms are comparable on a weight basis, the amino acid compositions are quite similar, and the N-terminal sequences are nearly identical for 24 residues. These similarities suggest a possible precursor-product relationship between the two carboxylester lipase forms.     

Bacterial adsorption to thermoresponsive polymer surfaces

The ability of microorganisms to `recognise' a change in the hydrophobicity/hydrophilicity balance of the surface was demonstrated using thermoresponsive poly(N-isopropylacrylamide) co-polymers with different Lower Critical Solution Temperatures. The polymers were grafted onto hydrolysed glass under well controlled conditions and the adhesion was followed using ¹³C-labelled Listeria monocytogenes. Attachment of the bacteria was found to be directly affected by the polymer transition from a hydrophilic to a hydrophobic state but by less than one order of magnitude.     

CO2 adsorption on polar surfaces of ZnO

Physical and chemical adsorption of CO₂ on ZnO surfaces were studied by means of two different implementations of periodic density functional theory. Adsorption energies were computed and compared to values in the literature. In particular, it was found that the calculated equilibrium structure and internuclear distances are in agreement with previous work. CO₂ adsorption was analyzed by inspection of the density of states and electron localization function. Valence bands, band gap and final states of adsorbed CO₂ were investigated and the effect of atomic displacements analyzed. The partial density of states (PDOS) of chemical adsorption of CO₂ on the ZnO(0001) surface show that the p orbitals of CO₂ were mixed with the ZnO valence band state appearing at the top of the valence band and in regions of low-energy conduction band.

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

The interaction between human pancreatic carboxylester hydrolase (bile-salt-stimulated lipase of human milk) and lactoferrin

An interaction between lactoferrin and human pancreatic carboxylester hydrolase (carboxylic-ester hydrolase, EC 3.1.1.1) (of bile-salt-stimulated lipase from human milk) has been demonstrated using partition in an aqueous two-phase system. This binding was strongly increased by the presence of sodium taurocholate, giving an apparent dissociation constant of around 10(-7) M. With this constant, significant binding is expected to occur in the intestine of the newborn being breast-fed between lactoferrin and either the pancreatic carboxylester hydrolase or the milk bile-salt-stimulated lipase. For carboxylester hydrolase, the interaction with lactoferrin meant a 1.4-fold increase in hydrolytic activity against p-nitrophenylacetate and cholesterololeate. For the function of lactoferrin we have not studied the importance of this interaction.     

Oriented adsorption of purple membrane to cationic surfaces

We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine- treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze- fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Nonspecific high affinity binding of bile salts to carboxylester lipases

The interactions with bile salts of carboxylester lipases (EC 3.1.1.13) from human pancreatic juice and pig pancreas were characterized by physical methods. Bile salts cause a decrease in the fluorescence intensity of the proteins at the emission maximum of 333-335 nm. The concentration dependence of this decrease shows saturation behavior, is relatively nonspecific with respect to bile salt conjugation or the presence of the 7 alpha-hydroxyl group, and is consistent with a 1:1 interaction between enzyme and bile salt. Direct measurement of the binding of [3H]cholate by equilibrium dialysis supports the stoichiometry. Other detergents also bind, causing fluorescence changes, but with much lower affinities. Binding of taurocholate to the monomeric pig enzyme is enhanced by increasing ionic strength, indicating the predominance of hydrophobic interactions. In the range of pH 5.5-6.8, binding is pH-independent with dissociation constants of 3-20 microM. At higher pH, affinity is greatly reduced and the fluorescence spectrum changes, indicating the importance of a protonated group for efficient interaction. Occupancy of the bile salt binding site partially stabilizes the enzyme against inactivation by heat but not trypsin. However, circular dichroism spectra do not indicate that bile salt binding is accompanied by any change in secondary structure. The monomeric pig enzyme binds to the argon/water interface in the presence of bile salts and binding of taurocholate to diisopropylphosphoryl-enzyme is similar to that measured with native enzyme. These results suggest that surface binding and catalysis occur at sites distinct from the bile salt binding site of the enzyme. Stabilization of the monomeric pig enzyme against denaturation at high energy surfaces occurs concomitantly with occupancy of the bile salt binding site. Overall, the data suggest that an important role of bile salts in vivo is to stabilize these enzymes at lipid-water interfaces.     

Substrate specificity of Geotrichum candidum lipase preparations

Summary Lipases with different fatty acid specificity were produced byGeotrichum candidum depending on growth condition. The hydrolysis of olive oil was inhibited by glycerol tributyrate and was dependent on Ca-ions for running at maximal rate.     

Effect of lipase specificity on triglyceride synthesis

Summary Enzymatic triglyceride synthesis from free fatty acid and glycerol is shown to be catalysed by both 1,3-specific lipase and non-specific lipase. This paper elucidates the mechanism of the reaction, showing that, with a 1,3-specific lipase, the 1,3-diolein enzymatically formed has to isomerise spontaneously to 1,2-diolein in presence of oleic acid, before being transformed to triolein by the enzyme.