Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: assessment by chemical modification

The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 mM. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor. Phenylmethanesulfonyl fluoride, another serine-specific reagent, did not inhibit either hydrolysis or transacylation. Therefore, we suggest that DFP does not modify an active serine in the catalytic site. p-Hydroxymercury benzoate and N-ethylmaleimide (NEM) abolished both activities of the enzyme. The presence of substrate partially protected against inactivation. Far-uv CD spectrum of NEM-modified enzyme revealed no changes in protein structure. The existence of two classes of essential cysteine residues was deduced from kinetics of NEM inactivation. Both classes differ in NEM reactivity and also in their participation in the catalytic mechanism. A tyrosine-specific reagent, tetranitromethane, also inhibited hydrolysis and transacylation, following first-order kinetics. The partial protection by substrate suggested the possible existence of essential tyrosines near the active site. At pH 5.0 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline inactivated hydrolysis but not transacylation. However, both of them remained unchanged at pH 6.5. The substrate prevented the loss of hydrolytic ability. Therefore, a carboxyl residue participating just in the catalytic mechanism of hydrolysis is proposed.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Evidence of a pH-dependent conformational change at the active site of lysolecithin:lysolecithin acyltransferase from rabbit lung

It has been shown that both activities, hydrolysis and transacylation, of lysolecithin:lysolecithin acyltransferase, as well as the conformation of the polypeptide are critically dependent on a pK around 5.8, but the question remains if the same residue(s) is responsible for the conformational change and the loss of activity. In this paper, ultrasonic cavitation is used to study the pH-dependent inactivation. The results show that there are two first-order inactivation constants which depend on pH and that the transition between them has a pK of 5.9. As the constants of ultrasonic inactivation are very dependent on the accessibility of the residues it is concluded that the conformational change modifies the accessibility of the active site.     

Thrombin-induced transfer of arachidonic acid in human platelets is not inhibited by trifluoperazine

Human platelets have been shown to contain a Ca++- and CoA-independent transacylase enzyme that catalyzes the transfer of arachidonic acid from phosphatidylcholine (PC) to lysoplasmenylethanolamine. It has been suggested that this route may represent a major source for released arachidonic acid in stimulated platelets. In this study, we have shown using arachidonic-labelled human platelets that the thrombin-induced activation of a transacylase reaction was not affected by concentrations of trifluoperazine (TFP) (15 micrograms/2 X 10(9) cells) which abolished the accumulation of free [3H]arachidonic acid in the presence of the cyclooxygenase/lipoxygenase inhibitor BW755C. TFP, at this concentration failed to block the hydrolysis of phosphatidylcholine (PC) completely and had no effect on the increased radioactivity seen in total phosphatidylethanolamine (PE) (160% of control after 4 min of incubation). These results suggest that the transacylase pathway activated in response to thrombin is not likely dependent on calcium. As TFP blocks effectively both the accumulation of free [3H]arachidonic acid and the mass of arachidonic acid without affecting the transfer of this fatty acid from PC to PE in thrombin-stimulated human platelets, it is very unlikely that the transacylation pathway represents a major source of release arachidonic acid. Based on these findings, we conclude that the above pathway may be primarily involved in the turnover of plasmenylethanolamine lipids in stimulated human platelets.     

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9-6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non-specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Synthesis and characterization of betaine-like diacyl lipids: zwitterionic lipids with the cationic amine at the bilayer interface

We synthesized and characterized a series of zwitterionic, acetate-terminated, quaternized amine diacyl lipids (AQ). These lipids have an inverted headgroup orientation as compared to naturally occurring phosphatidylcholine (PC) lipids; the cationic group is anchored at the membrane interface, while the anionic group extends into the aqueous phase. AQ lipids preferentially interact with highly polarizable anions (ClO(4)(-)) over less polarizable ions (Cl(-)), in accord with the Hofmeister series, as measured by the change in zeta potential of AQ liposomes. Conversely, AQ lipids have a weaker association with calcium than do PC lipids. The transition temperatures (Tm) of the AQ lipids are similar to the Tm observed with phosphatidylethanolamine (PE) lipids of the same chain length. AQ lipids form large lipid sheets after heating and sonication; however, in the presence of cholesterol (Chol), these lipids form stable liposomes that encapsulate carboxyfluorescein. The AQ:Chol liposomes retain their contents in the presence of serum at 37°C, and when injected intravenously into mice, their organ biodistribution is similar to that observed with PC:Chol liposomes. AQ lipids demonstrate that modulating the headgroup charge orientation significantly alters the biophysical properties of liposomes. For the drug carrier field, these new materials provide a non-phosphate containing zwitterlipid for the production of lipid vesicles.     

Liposome/DNA systems: correlation between association, hydrophobicity and cell viability

Small unilamellar vesicles associated with plasmid DNA showed maximum association efficiency for a cationic mixture of egg phosphatidylcholine (EPC):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):di-1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) (16:8:1 molar ratio) [65%], followed by neutral lipids EPC:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):cholesterol (Chol) (2:2:1 molar ratio) [30%], and a polymerized formulation 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC):DMPE:Chol (2:2:1 molar ratio) [11%]. The hydrophobicity factor (HF) for these formulations followed the trend DC8,9PC:DMPE:CHOL < EPC:DMPE:Chol < EPC:DOPE DOTAP, and DNA association did not alter this trend. Results suggest that the higher the HF value, the more fluid the membrane and the higher the efficiency of DNA association. On the other hand, no differences were observed in cell toxicity with lipids up to 1 mg/ml in VERO cells.     

Cubic phases in phosphatidylcholine-cholesterol mixtures: cholesterol as membrane "fusogen"

X-ray diffraction reveals that mixtures of some unsaturated phosphatidylcholines (PCs) with cholesterol (Chol) readily form inverted bicontinuous cubic phases that are stable under physiological conditions. This effect was studied in most detail for dioleoyl PC/Chol mixtures with molar ratios of 1:1 and 3:7. Facile formation of Im3m and Pn3m phases with lattice constants of 30-50 nm and 25-30 nm, respectively, took place in phosphate-buffered saline, in sucrose solution, and in water near the temperature of the Lalpha-HII transition of the mixtures, as well as during cooling of the HII phase. Once formed, the cubic phases displayed an ability to supercool and replace the initial Lalpha phase over a broad range of physiological temperatures. Conversion into stable cubic phases was also observed for mixtures of Chol with dilinoleoyl PC but not for mixtures with palmitoyl-linoleoyl PC or palmitoyl-oleoyl PC, for which only transient cubic traces were recorded at elevated temperatures. A saturated, branched-chain PC, diphytanoyl PC, also displayed a cubic phase in mixture with Chol. Unlike the PEs, the membrane PCs are intrinsically nonfusogenic lipids: in excess water they only form lamellar phases and not any of the inverted phases on their own. Thus, the finding that Chol induces cubic phases in mixtures with unsaturated PCs may have important implications for its role in fusion. In ternary mixtures, saturated PCs and sphingomyelin are known to separate into liquid-ordered domains along with Chol. Our results thus suggest that unsaturated PCs, which are excluded from these domains, could form fusogenic domains with Chol. Such a dual role of Chol may explain the seemingly paradoxical ability of cell membranes to simultaneously form rigid, low-curvature raft-like patches while still being able to undergo facile membrane fusion.     

Stimulation of galactosyltransferase in liver microsomes by lysolecithin

Lysolecithin markedly stimulated membrane-bound UDP-galactose:glycoprotein galactosyltransferase. The parent molecule lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidic acid, lysophosphatidic acid or glycerophosphorylcholine did not activate the enzyme suggesting that both fatty acyl- and phosphorylcholine groups are required for the enzyme activation. The dose-effect of lysolecithin showed sigmoidal kinetics and the Vmax of the enzyme was increased several-fold by lysolecithin. Saturating amounts of Triton masked the effect of lysolecithin. Pre-incubation with phospholipase A also activated the enzyme. A possible role of membrane lysolecithin is indicated in regulating the enzymes of glycoprotein synthesis.     

Effects of composition and molecular packing on ionic properties of lipid monolayer having both cationic and anionic head groups

The effects of composition and molecular packing on the overall ionic property of mixed monolayer involving cationic, anionic and zwitterionic lipids were studied by measuring surface potential change when the concentration of sodium or calcium ions in the aqueous substrate was varied. Those lipids used were N,N-dimethyl-N-n-hexadecyl-n-octadecyl ammonium chloride (DDAC) as cationic lipid, stearic acid (StH) or sodium docosylsulfate (SDocS) as anionic lipid, and 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (PE) and 1,2-distearoyl-sn-glycero-3-phosphorylcholine (PC) as ampholytic lipids. For the equimolar mixture of StH and DDAC, the reversal of apparent charge is observed when molecular packing exceeds 3.25 X 10(-10) M/cm2. The effect is rendered to the discreteness of cationic and anionic charges in the monolayer. It was also found that the addition of 30% of PC drastically changes the ionic property of PE closer to that of PC.     

Prostaglandin E(2) and tumour necrosis factor-alpha release by monocytes are modulated by phospholipids

The regulation of pro- and anti-mediator release from cells within the alveolar space would represent a desirable mechanism serving to protect this delicate gas-exchanging region of the lung. This study investigates the effect of alveolar surfactant lipids on the regulation of tumour necrosis factor alpha (TNF-alpha), a potent inflammatory cytokine, and prostaglandin E(2)(PGE(2)), a lipid mediator with anti-inflammatory properties. The results of this investigation reveal a marked effect on the release of these two important mediators from a monocytic cell line, MonoMac 6 (MM6), by phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (Chol) and sphingomyelin (SM). PC, PE and Chol demonstrated marked downregulation of TNF-alpha production at lipid concentrations of 125 and 250 microg/ml. Interestingly, SM significantly up regulated the release of TNF-alpha at these concentrations. However, the release of PGE(2)in MM6 cells incubated with the same lipids was significantly increased with PC and Chol, and significantly decreased in cells pre-treated with SM. This indicates a role for these lipids in alveolar immunoregulation.     

Thermodynamic properties and characterization of proteoliposomes rich in microdomains carrying alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts", ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc=41.5°C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt(1/2) values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ?H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.     

Solubilization by lysolecithin and purification of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe.

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient. The specific activity of the Mg2+-requiring plasma membrane ATPase activity (EC 3.6.1.3) was enriched from 0.3 mumol min-1 x mg-1 of protein in the homogenate to 26 in the purified membranes. The optimal conditions for solubilization of the ATPase activity by lysolecithin were found to be: 2 mg/ml of lysolecithin, a lysolecithin to protein ratio of 8 at pH 7.5, and 15 degrees C in the presence of 1 mM ATP and 1 mM ethylenediaminetetraacetic acid. A 6- to 7-fold purification of the solubilized ATPase activity was obtained by centrifugation of the lysolecithin extract in sucrose gradient. Part of the ATPase activity which was inactivated during the centrifugation in the sucrose gradient could be restored by addition of a micellar solution of 50 microgram of lysolecithin/ml during the assay. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purified enzyme showed only one band of Mr = 105,000 stained with Coomassie blue. Another ATPase component of apparent molecular weight lower than 10,000 was stained by periodic Schiff reagent but not colored by Coomassie blue. The purified enzyme was 85% inhibited by 50 micrometer N,N'-dicyclohexylcarbodiimide and 94% inhibited by 53 microgram of Dio-9/ml.     

Influence of cholesterol on sphingomyelin metabolism and hemileaflet fluidity of rat liver plasma membranes.

The objective of this study was to examine the effect of dietary Chol supplementation on SM metabolism in rat liver plasma membranes, as well as on membrane leaflet fluidity characteristics. The membrane Chol content increased significantly during the first 20 days of dietary feeding, but returned to the level of the control group when the diet was continued for another ten days. The initially more fluid outer leaflet of the membrane rigidified as a result of the diet, obliterating the natural asymmetry in the fluidity of the membrane bilayer. Changes in the neutral SMase activity were also observed. These changes were in strong negative correlation (r = -0.978) with the Chol/Pr ratio and are consistent with the in vitro inhibition of SMase activity reported earlier. In contrast, the SM synthesizing enzymes, PC:Cer-PCh and PE:Cer-PEt transferase, were stimulated in course of the dietary Chol feeding. The activity of PC:Cer-PCh transferase was more strongly affected. Our results support the concept that SM metabolism is regulated coordinately with that of Chol. The present work could contribute to the better understanding of the parallel accumulation of SM and Chol observed in a variety of pathological conditions such as atherosclerosis and Niemann-Pick disease.     

The effect of dbcAMP on the lysolecithin induced hemolysis of calf and adult cattle erythrocytes.

The calf erythrocytes have an increased sensitivity against lysolecithin as compared to their adult counterparts. 10(-3)M dbcAMP increases the hemolysis induced by 5 microgram of lysolecithin in 0.15 M NaCl containing 10 mM phosphate buffer (pH 7.4). By increasing the level of phosphate buffer (75 mM) in the incubation mixture, 10(-3)M dbcAMP decreases the hemolysis induced by 5 microgram of lysolecithin. These data suggest a dual effect exerted by dcbAMP: the relatively labilizing or stabilizing effect prevails as a function of exogenous inorganic phosphate level. 10(-6)M dcbAMP also has a relative protective effect against lysolecithin. The combined addition of cAMP (10(-3)) and theophyllin (10(-4)M) does not stabilize the membrane. By increasing the level of lysolecithin to 20 microgram/ml the stabilizing effect of dcbAMP disappears. DbcAMP (10(-3)) as well as cAMP (10(-3)M) and theophyllin (10(-4)M) have a minimum increasing effect on hemolysis in the absence of lysolecithin, too.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.