Action of phospholipase A2 on bilayers. Effect of inhibitors

Action of several solutes on the kinetics of phospholipase-A2-catalyzed hydrolysis of the ternary codispersions containing dimyristoylphosphatidylcholine + 1-palmitoyllysophosphatidylcholine + palmitic acid is examined. The kinetics of hydrolysis is interpreted in terms of the ability of the enzyme to bind to the substrate interface. The inhibitory effect of these solutes is correlated with their ability to modify fluorescence intensity of the bound enzyme, to modify the phase-transition profile, and to inhibit aggregation/fusion of the ternary codispersions. Based on these observations, it is suggested that the solutes like n-alkanols, ketamine, alphadolone, alphaxalone, flufenamic acid, tobramycin, mepacrine, EMD 21657 and U-10029A modulate the phase equilibria in the codispersions and thus noncompetitively inhibit the phospholipase action. Inhibition by feverfew extract (Tanacetum parthemium) is also by a similar mechanism. Lipid-soluble drugs as indomethacin had little effect on the kinetics of hydrolysis. All these inhibitors decrease the total extent of hydrolysis of the available substrate. However, none of these inhibitors have any effect on the hydrolysis of monomeric substrate or on the inactivation of the phospholipase A2 by p-bromophenacylbromide. These observations suggest that all these inhibitors do not interact directly with the catalytic site of the free or the bound enzyme, and their effect is primarily on the enzyme-binding sites on the substrate vesicle, that is, by perturbation of lipid-protein interaction.     

Interaction of phospholipase A2 and phospholipid bilayers

Binding of phospholipase A₂ from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC₁₄) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC₁₄ alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of the pig-pancreatic enzyme to vesicles of the diether-PC₁₄ could not be detected even after long incubation (up to 24 h) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.     

Action of phospholipase A2 on bilayers. Effect of fatty acid and lysophospholipid additives on the kinetic parameters

Action of pig pancreatic phospholipase A2 on the ternary codispersions of diacylphosphatidylcholine, 1-acyllysophosphatidylcholine and fatty acids is examined. The binding and kinetic constants are found to be the same under a variety of conditions. These parameters and the catalytic turnover number change with the phase-transition temperature of the ternary codispersions, and optimal binding, kinetic and catalytic constants are seen in the phase-transition range where an equilibrium exists between laterally separated phases. The effect of changing the structure of any of the three components is also via a change in the phase-transition temperature of their ternary codispersions. These observations suggest that the binding of pig pancreatic phospholipase A2 to the defect sites on the substrate interface determines the substrate concentration dependence of the initial rate of hydrolysis, and the catalytic turnover by the bound enzyme also depends upon the phase state of the bilayer. An additive-induced stabilization of the defects in the substrate bilayer is postulated to account for the enhanced binding of the enzyme to the bilayer.     

Influence of product phase separation on phospholipase A(2) hydrolysis of supported phospholipid bilayers studied by force microscopy

In situ atomic force microscopy studies reveal a marked influence of the initial presence of hydrolysis products on the hydrolysis of supported phospholipid bilayers by phospholipase A(2). By analysis of the nano-scale topography of a number of supported bilayers with different initial product concentrations, made by Langmuir-Blodgett deposition, we show that small depressions enriched in products are efficiently promoting enzyme degradation of the bilayer. These small depressions, which are indicative of phase separation, are initially present in samples with 75% products. The kinetics of phospholipase A(2) exhibit under certain conditions an initial phase of slow hydrolysis, termed the latency phase, followed by a marked increase in the hydrolysis rate. The appearance of the phase-separated bilayer is strikingly similar to that of bilayers at the end of the latency phase. By analysis of individual nano-scale defects we illustrate a quantitative difference in the growth rates of defects caused by product aggregation and other structural defects. This difference shows for the first time how the enzyme prefers one type of defect to another.     

Interaction of melittin and phospholipase A2 with azobenzene-containing phospholipid

Interactions of melittin and/or phospholipase A2 (PLA2) with circular dichroism (CD)-active phospholipid, bis(4'-n-octanoxyazobenzene-4-carboxyl)-L-alpha-phosphatidylcholin e (CDPC), were studied. In the presence of melittin at a lipid-to-melittin molar ratio (Ri) of 5, multilamellar dispersion, composed of CDPC and dipalmitoylphosphatidylcholine with a molar ratio of 1, underwent morphological change to form small melittin-lipid particles. When PLA2 was added to the melittin-lipid particles at 37 degrees C, the CD band at 222 nm exhibited a remarkable enhancement depending on Ri, indicating the formation of melittin-PLA2-lipid complex. After a 30 min incubation of melittin-PLA2-lipid complex at 45 degrees C in the presence of Ca2+, the CD band at 222 nm was still enhanced and a new positive band at 356 nm was observed. On the other hand, in the absence of Ca2+, the CD enhancement characteristic of melittin-PLA2-lipid complex disappeared after the incubation at 45 degrees C. These results suggest that the melittin-PLA2-lipid complex did not undergo any drastic morphological change upon PLA2-catalyzed hydrolysis of lipid, and that Ca2+ is indispensable in order that the melittin-PLA2-lipid complex remains intact and PLA2 exerts efficient hydrolytic activity in the melittin-PLA2-lipid complex.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers

The free energy of unfolding of a membrane protein from lipids into water (ΔG^o_w,l) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein's sequence-structure-energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe the experimental process that allowed us to measure the ΔG^o_w,l of the outer membrane phospholipase A into large unilamellar vesicles (LUVs) of 1,2-dilauroyl-sn-glycero-3-phosphocholine. To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition and liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary “holdase” to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure ΔG^o_w,l for other membrane proteins.     

The effect of gramicidin A on phospholipid bilayers

The helical polypeptide, gramicidin A has been widely studied as a model for the interactions of hydrophobic proteins with lipid bilayer membranes. Many reports are now available of the physical effects of mixing gramicidin A with phospholipid membranes, however, the interpretation of these data remains unclear. The purpose of this communication is to examine the controversial claim that high concentrations of gramicidin A cause disorder within the L phase of phosphatidylcholine-water dispersions. Solid-state nuclear magnetic resonance (NMR), density gradient and X-ray diffraction techniques are used to confirm the existence of such an effect and mechanisms are discussed which account for the known effects of gramicidin A on lipid bilayers.     

Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.

In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in a membrane and to investigate the activation process. Three single-cysteine variant proteins H26C, H234C, and S144C were produced and purified to homogeneity. Using maleimido-based homo-bifunctional cross-linking reagents, H26C could be efficiently cross-linked as assessed by SDS-PAGE, whereas S144C and H234C could not be cross-linked. These data suggest that residue 26 is located close to the dimer symmetry axis. H26C was specifically labeled with 5-({[(2-iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid and N,N'-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine as the fluorescent energy donor and acceptor, respectively, and dimerization was investigated using fluorescence resonance energy transfer (FRET). Quenching of the donor in the presence of the acceptor demonstrated the dimeric nature of OMPLA, in agreement with cross-linking data. The observed FRET effect was dependent on the cofactor calcium, and the presence of substrate, indicating the specificity of the dimerization process. The labeled protein was reconstituted in phospholipid vesicles. In bilayers, OMPLA exhibited low activity and was dimeric as assessed by FRET. Addition of detergent resulted in a 70-fold increase in activity, while the protein remained dimeric. The results are discussed in terms of the activation of dimeric OMPLA due to changes in the physical state of the bilayer which occur upon perturbation of the membrane integrity.     

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2.

We have investigated the time course of the degradation of a supported dipalmitoylphosphatidylcholine bilayer by phospholipase A2 in aqueous buffer with an atomic force microscope. Contact mode imaging allows visualization of enzyme activity on the substrate with a lateral resolution of less than 10 nm. Detailed analysis of the micrographs reveals a dependence of enzyme activity on the phospholipid organization and orientation in the bilayer. These experiments suggest that it is possible to observe single enzymes at work in small channels, which are created by the hydrolysis of membrane phospholipids. Indeed, the measured rate of hydrolysis of phospholipids corresponds very well with the enzyme activity found in kinetic studies. It was also possible to correlate the number of enzymes at the surface, as calculated from the binding constant to the number of starting points of the hydrolysis. In addition, the width of the channels was found to be comparable to the diameter of a single phospholipase A2 and thus further supports the single-enzyme hypothesis.     

Effect of N-methylation of phosphatidylethanolamine on the fluidity of phospholipid bilayers

The effect of N-methylphosphatidylethanolamine on phase transition and the fluidity of the liposomes made of dipalmitoylphosphatidylcholine or phosphatidylethanolamine was studied by the steady-state fluorescence polarization method and differential scanning calorimetry. N-methylation of phosphatidylethanolamine caused a decrease of fluidity of liposomes made of dipalmitoylphosphatidylcholine, but had little effect on dipalmitoylphosphatidylethanolamine. The liposomes prepared with both phosphatidylcholine and N-methylphosphatidylethanolamine and also phosphatidylethanolamine and N-methylphosphatidylethanolamine could be composed of solid solution and exhibited symmetric phase diagram.     

Effect of transbilayer phospholipid distribution on erythrocyte fusion

Phospholipid packing has been suggested as a relevant variable in the control of membrane fusion events. To test this possibility in a model system, a comparison was made of the fusability of erythrocytes with a normal asymmetric transbilayer distribution of plasma membrane phospholipids (tightly packed exterior lipids) and erythrocytes with a symmetric transbilayer distribution of phospholipids (more loosely packed exterior lipids), using polyethylene glycol as fusogen. Not only were lipid-symmetric cells more readily fused, but fusions of mixtures of lipid-symmetric and lipid-asymmetric cells indicated that both fusing partners must have a symmetric distribution for fusion to be enhanced. Lipid-symmetric cells may fuse more readily because loose packing of the exterior lipids enhances hydrophobic interactions between cells. Alternatively, enhanced membrane fluidity may facilitate intramembranous particle clustering, previously implicated as a potentiator of fusion. Finally, exposure of phosphatidylserine on the surface of lipid-symmetric erythrocytes may be responsible for their enhanced fusion.     

Properties of unsaturated phospholipid bilayers: Effect of cholesterol

Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.     

Effect of free cholesterol on incorporation of triolein in phospholipid bilayers

Triacylglycerols are the major substrates for lipolytic enzymes that act at the surface of emulsion-like particles such as triglyceride-rich lipoproteins, chylomicrons, and intracellular lipid droplets. This study examines the effect of cholesterol on the solubility of a triacylglycerol, triolein, in phospholipid surfaces. Solubilities of [carbonyl-13C]triolein in phospholipid bilayer vesicles containing between 0 and 50 mol % free cholesterol, prepared by cosonication, were measured by 13C NMR. The carbonyl resonances from bilayer-incorporated triglyceride were shifted downfield in the 13C NMR spectra from those corresponding to excess, nonincorporated material. This enabled solubilities to be determined directly from carbonyl peak intensities at most cholesterol concentrations. The bilayer solubility of triolein was inversely proportional to the cholesterol/phospholipid mole ratio. In pure phospholipid vesicles the triolein solubility was 2.2 mol %. The triglyceride incorporation decreased to 1.1 mol % at a cholesterol/phospholipid mole ratio of 0.5, and at a mole ratio of 1.0 for the bilayer lipids, the triolein solubility was reduced to just 0.15 mol %. The effects of free cholesterol were more pronounced and progressive than observed previously on the bilayer solubility of cholesteryl oleate (Spooner, P. J. R., Hamilton, J. A., Gantz, D. L., & Small, D. M. (1986) Biochim. Biophys. Acta 860, 345-353]. As with cholesteryl oleate, we suggest that cholesterol also displaces solubilized triglyceride to deeper regions of the bilayer.     

Effect of cholesterol on the structure and dynamic properties of unsaturated phospholipid bilayers

Molecular dynamics (MD) computer simulations of five different hydrated unsaturated phosphatidylcholine lipid bilayers built up by 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules with 40 mol% cholesterol, and the same five pure phosphatidylcholine bilayers have been performed at 303 K. The simulation box of a lipid bilayer contained 96 phosphatidylcholines, 64 cholesterols, and 3840 water molecules (48 phosphatidylcholine molecules and 32 cholesterols per layer and 24 water molecules per phospholipid or cholesterol in each case). The lateral self-diffusion coefficients of the lipids in these systems and mass density profiles with respect to the bilayer normal have been analyzed. It has been found that the lateral diffusion coefficients of phosphatidylcholine molecules increase with increasing number of double bonds in one of the lipid chains, both in pure bilayers and in bilayers with cholesterol. It has been found as well that the lateral diffusion coefficient of phosphatidylcholine molecules of a lipid bilayer with 40 mol% cholesterol is smaller than that for the corresponding pure phosphatidylcholine bilayer.