Surface pressure-dependent interactions of secretory phospholipase A2 with zwitterionic phospholipid membranes

The hydrolytic activity of secretory phospholipase A(2) (PLA(2)) is regulated by many factors, including the physical state of substrate aggregates and the chemical nature of phospholipid molecules. In order to achieve strong binding of PLA(2) on its substrates, many previous works have used anionic lipid dispersion to characterize the orientation and penetration depth of PLA(2) molecules on membrane surfaces. In this study, we applied monolayer technique with controllable surface area to investigate the PLA(2)s of Taiwan cobra venom and bee venom on zwitterionic phophatidylcholine monolayers and demonstrated an optimum hydrolytic activity at a surface pressure of 18 and 24 mN/m, respectively. By combining polarized attenuated total reflection Fourier-transform infrared spectroscopy and monolayer-binding experiments, we found that the amount of membrane-bound PLA(2) decreased markedly as the surface pressure of the monolayer was increased. Interestingly, the insertion area of the PLA(2)s decreased to near zero as the surface pressure increased to the optimum pressure for hydrolytic activity. On the basis of the measured infrared dichroic ratio, the orientation of the PLA(2)s bound to zwitterionic membranes was similar to that observed on a negatively charged membrane and was independent of the surface pressure. Our findings suggest that both PLA(2)s were located on the membrane surface rather than penetrating the membrane bilayer and that the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes. The results are discussed in terms of the easy access of catalytic water for the PLA(2) activity and the mobilization of its substrate and product to facilitate the catalytic process.     

Enzymatic hydrolysis reaction of phospholipids in monolayers

The hydrolysis reaction of , and , -dipalmitoylphosphatidylcholine (DPPC) catalized by bee venom phospholipase A₂ was studied in spreading monolayer at the water/air interface. DPPC and the hydrolysis products, palmitic acid and -lysophosphatidylcholine, palmitoyl were characterized at the interface by means of surface pressure, surface potential and ellipsometric measurements. Furthermore, mixed monolayers of reagents and products were investigated to ascertain their miscibility. The results show that the hydrolysis reaction can be followed by the decrease of surface pressure with time on subphases containing β-cyclodextrin, a well-known complexing agent of many amphiphilic compounds. The order of the reaction, the kinetic constant and other kinetic parameters are deduced.     

Reorganization of lipid nanocapsules at air-water interface 3. Action of hydrolytic enzymes HLL and pancreatic PLA2

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A2 (PLA2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac, Solutol and Lipoid, is studied by simultaneous measuring the changes in the film area and the surface potential in the "zero order" trough at constant surface pressure (pi). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.     

Comparative study of lipolysis by PLA2 of DOPC substrates organized as monolayers, bilayer vesicles and nanocapsules

The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. One biologically relevant example is the enzymatic hydrolysis of DOPC by PLA(2), which results in cleavage of phospholipids molecules into water insoluble lipolytic products, namely oleic acid and lysophospholipid. In general, the enzymatic activity depends on the substrate organization and molecular environment of the catalytic reaction. The lipolysis by phospholipase A(2) of dioleoylphosphatidylcholine substrates organized as monolayer, bilayers vesicles and lipid nanocapsules was studied by measuring the decrease of the surface area at constant surface pressure or increase of the surface pressure at constant area at air-water interface. A kinetic model describing the coupling of the catalytic act with corresponding interfacial phenomena was developed. By using the kinetic model the values for the global hydrolytic kinetic constants were obtained. The obtained value for the monolayer is five orders of magnitude higher than this obtained with small unilamellar vesicles and six orders of magnitude higher then those obtained with lipid nanocapsules. The comparison shows that the enzymatic catalytic act occurring in the lipid environment of the monolayer is more efficacious than at the vesicle and nanocapsules interfaces.     

Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis

The activity of phospholipase A(2) (PLA(2)) which catalyzes the hydrolysis of phospholipids into free fatty acids and lysolipids, depends on the structure and thermodynamic state of the membrane. To further understand how the substrate conformation correlates with enzyme activity, model systems that are based on time-resolved membrane microscopy are needed. We demonstrate a methodology for preparing and investigating the dynamics of fluid supported phospholipid membranes hydrolyzed by snake venom PLA(2). The method uses quantitative analysis of time-lapse fluorescence images recording the evolution of fluid bilayer islands during hydrolysis. In order to minimize interactions with the support surface, we use double bilayer islands situated on top of a complete primary supported membrane prepared by hydration of spincoated lipid films. Our minimal kinetic analysis describes adsorption of enzyme to the membrane in terms of the Langmuir isotherm as well as enzyme kinetics. We use two related models assuming hydrolysis to occur either at the perimeter or at the surface of the membrane island. We find that the adsorption constant is similar for the two cases, while the estimated turnover rate is markedly different. The PLA(2) concentration series is measured in the absence and presence of beta-cyclodextrin which forms water soluble complexes with the reaction products. The results demonstrate the versatility of double bilayer islands as a membrane model system and introduces a new method for quantifying the kinetics of lipase activity on membranes by directly monitoring the evolution in substrate morphology.     

Laser desorption/ionization mass spectrometric assay for phospholipase activity based on graphene oxide/carbon nanotube double-layer films

A new method for quantitative phospholipase activity assays using mass spectrometry (MS) and a supported thin film consisting of a graphene oxide (GO)/carbon nanotube (CNT) double layer as a substrate for laser desorption ionization (LDI) has been developed. Phospholipids were very efficiently analyzed by LDI-time-of-flight (TOF) MS on the GO/CNT films, presumably because of the affinity of phospholipids for the GO/CNT surface. Therefore, the rate of lipid hydrolysis was conveniently measured using LDI-TOF mass spectra obtained from a GO/CNT surface on which the phospholipid hydrolysis reaction mixtures had been spotted by comparing the mass-peak intensities of reactants and products. The present platform for phospholipase assays based on MS and GO/CNT double-layer films enables quantitative measurements at low cost, allows assays to be performed in a short time, and is compatible with an array format, unlike conventional assay methods.     

具有抗炎、抗过敏活性磷脂酶抑制剂的研究进展

磷脂酶参与细胞跨膜信息传递, 磷脂酶A2 还是机体炎症、过敏介质产生的关键酶。因此, 磷脂酶抑制剂的合成研究对研究细胞表达某种功能的信息传递机制及与磷脂酶A2 激活相关的疾病治疗机制和药物研究具有重要意义。本文主要综述了近期有关磷脂酶A2 抑制剂的合成、结构、抑效性能、构效关系及应用前景等, 对磷脂酶C和磷脂酶D的抑制剂作了简要介绍。     

Fluorophore-encapsulated solid-supported bilayer vesicles: a method for studying membrane permeation processes

This letter describes a new method for studying the interaction of the membrane-lysing enzyme phospholipase A(2) (PLA(2)) with phospholipid bilayers by simultaneous measurements of enzyme binding and vesicle lysis using surface plasmon resonance (SPR) and permeabilization using surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The PLA(2) inhibitor dimethyl-eicosadienoic acid was incorporated into the surface-bound vesicles and support bilayer in order to study its role in preventing PLA(2)-mediated vesicle lysis. This methodology has a generic applicability for the study of a range of membrane-disrupting agents.     

Phospholipase A2 hydrolysis of mixed phospholipid vesicles formed on polyelectrolyte hollow capsules

Mixtures of the phospholipids L-alpha-dimyristoylphosphatidic acid (DMPA) and L-alpha-dipalmitoylphosphatidylcholine (DPPC) have been successfully adsorbed onto the charged surface of multilayer polyelectrolyte capsules to form a novel vesicle. Leaving such vesicles in phospholipase A(2) solution, we observed the hydrolysis reaction on the surface of the lipid/polymer vesicles and a permeability change before and after the reaction by confocal-laser scanning microscopy (CLSM). A capsule with adjustable permeability was constructed. This method may provide new features for drug-release vesicles.     

A Light‐Activated Microheater for the Remote Control of Enzymatic Catalysis

The remote control of enzymatic catalysis is of significant importance in disease treatment and industrial applications. Herein, we designed a microheater composed of a porous polylactic acid (PLA) matrix and polydopamine (PDA) with notable photothermal conversion capability. Starch hydrolysis, catalyzed by using α‐amylase, was accelerated in the presence of the microheater under illumination with near‐infrared light or natural sunlight at room temperature. Additionally, the methodology was extended to the preparation of microwave‐absorbing materials with the deposition of polyaniline on porous PLA matrix. The porous morphology improves the energy‐conversion efficiency.     

Action of Humicola lanuginosa lipase on mixed monomolecular films of tricaprylin and polyethylene glycol stearate

The hydrolysis catalyzed by Humicola lanuginosa lipase (HLL) of pure tricaprylin (TC) or stearate of polyethylene glycol 1500 (PEG-St) as well as their mixtures spread as monomolecular films were studied. The catalytic transformation of the two substrates TC or PEG-St into their respective reaction products was detected by measuring simultaneously the decrease in the film area and the surface potential using the "zero order" trough at constant surface pressure. A kinetic model describing the enzymatic hydrolysis was developed. The surface concentrations of the two substrates and their respective reaction products as well as the values of the global kinetic constants of hydrolysis were determined. The experimentally obtained global kinetic constants of the catalytic action of HLL against TC and PEG-St present in mixed monolayers of TC/PEG-St are approximately the same as in the case of pure monolayers. These obtained results give some indications that the activity of enzyme is not significantly affected by the different molecular environments in the mixed monolayers.     

Influence of dipalmitoylphosphatidylcholine (or dioleoylphosphatidylcholine) and phospholipase A2 enzyme on the properties of emulsions

The properties of n-tetradecane emulsions with dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) in 1M ethanol were investigated at 20 and 37°C. The zwitterionic phospholipids having the same headgroup bound to the apolar tail composed of two saturated or unsaturated chains were used as stabilizing agents. Both phospholipids may self-organize into aggregates, which possess different sizes and surface affinities. Electrokinetic properties of the systems at natural pH or pH 8 were investigated taking into account the effective diameter of the droplets as well as the zeta potentials using the dynamic light scattering technique. The effect of both phospholipids decreases the initially negative zeta potential of the n-tetradecane emulsion and is more evident in the case of DPPC especially at a physiological temperature near its main temperature transition. The change of zeta potential by DOPC is visible at both temperatures probably as an effect of a loose packing of this phospholipid on n-tetradecane droplets, because of the presence of double bonds in its molecule. Also, the role of ethanol dipoles on the stability of oil/phospholipid emulsions is obvious. The other aim of paper was the characterization of the phospholipase A(2) influence on DOPC hydrolysis in the emulsion environment in order to emphasize the importance of such methodology. The present work is the first study that explores the effects of both electrolyte ions and ethanol molecules on DOPC hydrolysis by phospholipase. The effect of enzyme on the n-tetradecane/DOPC emulsions was investigated at pH 8 with Na(+) or Ca(2+) ions, which occur in the physiological fluids. The effective diameters do not always correlate with the zeta potentials. A possible reason of such behavior might a mechanism different from the electrostatic stabilization. The particular role of Ca(2+) ions in the emulsions with phospholipids was confirmed. Those investigations provide insight into the properties of the PLA(2) hydrolysis process enhanced by added ethanol. It is believed that the enzyme effect on the phospholipid aggregation behavior at the oil-water interface will be helpful for understanding other biological phenomena.     

Structuring of supported hybrid phospholipid bilayers on electrodes with phospholipase A2

The effect of a lipolytic enzyme, pork pancreatic phospholipase A(2), on hybrid bilayer membranes was monitored using voltammetry, impedance spectroscopy and surface plasmon resonance. The hybrid bilayers were prepared by Langmuir-Schaefer transfer of lipid monolayers onto gold electrodes modified with self-assembled alkanethiol monolayers, or by liposome spreading. The electrodes were immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and cleavage of the ester bond in the sn-2 position of phospholipids in the outer leaflet of the hybrid layers. The action of phospholipase A(2) led to perforation of the lipid films. Impedance spectroscopy and surface plasmon resonance were used for monitoring enzyme adsorption, phospholipid hydrolysis and product desorption. The results obtained show that transport efficiency of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the bilayer depends on the action of the enzyme; the state of the lipid layer covering the electrode surface depends on the latter as well. Cyclic voltammetry and electrochemical impedance spectroscopy were used to study this effect. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using a bare gold electrode indicated facilitated penetration of the drug into the layer. The results obtained were interpreted in terms of pore formation in the lipid matrix; phospholipase A(2) can be considered as a nano-device for high precision perforation of the lipid layer.     

Enantiospecific synthesis of the phospholipase A2 inhibitor (-)-cinatrin B

The first enantiospecific synthesis of phospholipase A2 (PLA2) inhibitor (-)-cinatrin B (2) from the D-arabinose derivative 9 is described. The spirolactone system was formed by an Ireland-Claisen rearrangement of the allyl ester 8 followed by hydrolysis and stereoselective iodolactonization. The stereoselectivity of the rearrangement was controlled by the asymmetry in the allylic alcohol fragment. Ester (S)-8 gave the desired rearrangement product 7 and the epimer 13 in high yield as a 73:27 ratio, respectively. The final stereocenter at C2 was introduced via a chelation-controlled addition of the Grignard reagent derived from trimethylsilylacetylene to alpha-hydroxy ketone 6. Transformation of the terminal alkyne into the methyl ester 21 followed by acetal hydrolysis and selective lactol oxidation afforded cinatrin B methyl ester (22). Base hydrolysis and acid-induced relactonization then gave (-)-cinatrin B (2).     

The interaction between OPH and paraoxon at the air-water interface studied by AFM and epifluorescence microscopies

The paraoxon hydrolysis reaction catalyzed by organophosphorus hydrolase (OPH) monolayer at the air-water interface was studied. OPH-paraoxon interactions, occurring at the two-dimensional interface, by close-packed, highly orientated OPH monolayer, were investigated by several different surface chemistry techniques; e.g. surface pressure area isotherms, atomic force microscopy (AFM), and in situ epifluorescence microscopy. The characterization of OPH Langmuir and Langmuir-Blodgett films prepared in both the presence and absence of paraoxon, demonstrated significantly distinctive feature when compared with one another. Continuous growth of the OPH aggregates is a distinct phenomenon associated with hydrolysis, in addition to the pH changes in the local environment of the enzyme macromolecules.     

Enzyme activity to augment the characterization of tethered bilayer membranes

The rate of Ca2+ -triggered phospholipase A2 (PLA2) degradation of tethered bilayer membranes (tBLMs), composed of a synthetic lipid, beta-mercaptoethanol, and palmitoyloleoylphosphatidylcholine (POPC), is approximately 80 times greater than for those prepared with diphytanoylphosphatidylcholine (DPhyPC). Electrochemical impedance spectroscopy (EIS) and neutron reflectivity (NR) data indicate complete, water-free tBLMs that exhibit near ideal capacitive behavior and the presence of a water reservoir in the bilayer subspace proximal to the substrate (Au) surface for both tBLMs. Together these data indicate that the POPC and the DPhyPC tBLMs are structurally similar along the surface normal but markedly different at the outer leaflet/solution interface and that PLA2 is a sensitive probe of short length scale structural differences not revealed by EIS and NR.     

Intramolecular general base catalyzed ester hydrolysis. The hydrolysis of 2-aminobenzoate esters

Rate constants have been obtained for the hydrolysis of the trifluoroethyl, phenyl, and p-nitrophenyl esters of 2-aminobenzoic acid at 50 degrees C in H(2)O. The pseudo-first-order rate constants, k(obsd), are pH independent from pH 8 to pH 4 (the pK(a) of the amine group conjugate acid). The 2-aminobenzoate esters hydrolyze with similar rate constants in the pH-independent reactions, and these water reactions are approximately 2-fold slower in D(2)O than in H(2)O. The most likely mechanism involves intramolecular general base catalysis by the neighboring amine group. The rate enhancements in the pH-independent reaction in comparison with the pH-independent hydrolysis of the corresponding para substituted esters or the benzoate esters are 50-100-fold. In comparison with the hydroxide ion catalyzed reaction, the enhancement in k(obsd) at pH 4 with the phenyl ester is 10(5)-fold. Intramolecular general base catalyzed reactions are assessed in respect to their relative advantages and disadvantages in enzyme catalysis. A general base catalyzed reaction can be more rapid at low pH than a nucleophilic reaction that has a marked dependence on pH and the leaving group.     

Kinetics of spreading of DOPC liposomes at the air-water interface subjected to phospholipase A2 hydrolisis

The kinetics of surface film formation from DOPC small unilamellar liposomes spread at the air-water interface was studied by recording the surface pressure and the surface potential. The rate constants of the surface transformation of perfectly closed vesicles into open surface active structures was detcrmined.It was found that the surface transformation was accelerated by enzymatid hydrolysis. A theoretical approach, describing the coupling of the surface transformation with the catalytic hydrolysis in the scooting mode of bilayered liposomes is developed.Values of the specific activity of hydrolysis of DOPC bilayered vesicles by phospholipase A₂ fromVipera berus orientalis were obtained by this method and compared with those previously obtained by the classical pH-stat tirration method.     

Four orders of magnitude rate increase in artificial enzyme-catalyzed aryl glycoside hydrolysis

[reaction: see text] (6AR,6DR)-6A,6D-Di-C-cyano-beta-cyclodextrin (1) and 6A,6D-di-C-cyano-alpha-cyclodextrin (2) were synthesized and shown to catalyze hydrolysis of aryl glycosides into glucose and phenol with a reaction following Michaelis-Menten kinetics. At pH 8.0 and 59 degrees C hydrolysis of 4-nitrophenyl alpha-glucopyranoside was catalyzed by 1 with KM = 10.5 +/- 1.5 mM, kcat = 1.42(+/-0.09) x 10(-4) s(-1), and kcat/kuncat = 7922. Catalysis was observed with a concentration of 1 as low as 10 microM. Hydrolysis of the other aryl glycosides containing stereochemical variation in the sugar-moiety and 4-nitro-, 2-nitro-, 2-aldehydo-, and 2,4-dinitro- were also catalyzed by 1 and 2 with kcat/kuncat ranging from 4 to 7100. Hydrolysis of a phenyl beta-d-glucoside or the thioglycoside tolylthio beta-D-glucoside was also catalyzed. From a series of prepared analogues of 1 it was found that the catalysis was associated with the hydroxyl groups alpha to the nitril groups. The monocyanohydrin 6-C-cyano-beta-cyclodextrin (3) was also found to catalyze the hydrolysis of 4-nitrophenyl beta-glucopyranoside with kcat/kuncat = 1356. It was proposed that the cyclodextrin cyanohydrins 1-3 catalyze the hydrolysis by general acid catalysis on the bound substrate.     

Isotopic exchange of CO adsorbed on Pt(111)

The isotopic exchange of CO adsorbed on Pt(111) was studied using polarization modulation IR reflection absorption spectroscopy (PM-IRRAS) and temperature programmed desorption. It was found that the rate constants for the exchange reaction are much higher than would be expected from previous investigations of CO adsorbed on Pt nanoparticles. The adsorption of CO on Pt(111) under elevated pressures of CO and H(2) was also studied using PM-IRRAS. It was seen that CO pressures above 1 mbar lead to a shift in the absorption peak arising from CO adsorbed on a bridge site from 1850 to 1875 cm(-1). Exposing the CO-covered Pt(111) surface to 1000 mbar H(2) did not lead to any significant desorption of CO at room temperature, whereas at 363 K H(2) exposure did lead to a significant desorption of CO, due to the increased chemical potential of H(2). In a mixture of CO and H(2) with partial pressures of 0.01 mbar and 1000 mbar, respectively, no significant effect of H(2) on the PM-IRRAS spectrum was seen at temperatures below 423 K.