Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes.

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.     

Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization

We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location.     

Phospholipases A1 and A2 of rat liver plasma membranes; mechanism of action.

While V/S plots of phospholipase A1 show a phase transition, kinetic behaviour of phospholipase A2 acting in the same concentration range is hyperbolic. However after phospholipase A2 has been solubilized from the plasma membranes by 1 M NaCl, the V/S curve shows a phase transition. Membrane-bound phospholipase A1 shows a narrow optimum pH at 8.5 -9, while phospholipase A2 activity presents only small variations between pH 7 and 9.5. Towards exogenous phospholipids at the optimum pH 8.5 of phospholipase A1, the specific activity of the latter is 3-fold higher than phospholipase A2 specific activity. On the contrary towards endogenous phospholipids, phospolipase A2 activity is higher than phospholipase A2 activity. Moreover labeled endogenous PE hydrolysis by phospholipase A2 is decreased by addition of non labeled exogenous PE into the incubation medium. All these data suggest that the active site of phospholipase A1 is turned to the outside and acts only on exogenous substrates: for phospholipase A2 it would be inside, and exogenous phospholipids could be hydrolyzed only after penetrating the membrane.     

Acyl transfer reactions associated with cis Golgi apparatus of rat liver

Isolated Golgi apparatus, highly purified from rat liver, were found to contain an acyl transfer activity capable of restoring the acyl chains of the lysophospholipid products of the action of phospholipase A₂ on phosphatidylcholine. The activity was located primarily in cis and medial Golgi apparatus fractions, had a pH optimum of 6.0 to 7.5 and was stimulated by various acyl-CoA derivatives but not by fatty acids plus ATP. The activity, determined from the conversion of [¹⁴C]lysophosphatidylcholine to [¹⁴C]phosphatidylcholine, was unaffected by EGTA, inhibited by manoalide at high concentrations (0.2 mM), and temperature-dependent. Temperature dependency, however, showed no definite transition temperature over the range 15 to 37°C. The results demonstrated that cis Golgi apparatus membranes have the enzymatic capacity to restore fatty acids lost from phospholipids through the action of phospholipase A. The latter has been previously suggested to occur at the cis Golgi apparatus membranes based on analyses of cell-free transfer of radiolabeled phosphatidylcholine.     

Intracellular phospholipase activities of Tetrahymena pyriformis

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.     

Lysophospholipids in pig pancreatic zymogen granules in relation to exocytosis.

A hypothesis to explain the stimulatory role of cyclic AMP (adenosine 3':5'-monophosphate) in pancreatic enzyme secretion. has been tested. In this hypothesis cyclic AMP would activate a phospholipase activity, which would lead to a locally increased lysophospholipid formation, resulting in a fusion between the zymogen granule membrane and the apical plasma membrane. Cyclic AMP added to isolated pig pancreatic zymogen granules leads to an increased lysis of these granules, but the slowness of this effect makes its physiological significance dubious. In pancreatic homogenates or zymogen granules no stimulating effect of cyclic AMP on lipase of phospholipase activity could be demonstrated. Isolated zymogen granules have a high lysophospholipid content (27% of total phospholipids), consisting of the 1-acyl and 2-acyl forms of lysophosphatidylcholine and lysophosphatidylethanolamine. Experiments with radioactive phosphatidylcholine indicate that the lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure. It is concluded that these experiments do not lend support to the above hypothesis for the mechanism of action of cyclic AMP in pancreatic enzyme secretion     

Use of phospholipase A to compare phospholipid organization in synaptic membranes,myelin, and liposomes

Summary The pattern of fatty acid release from rat synaptic membranes in the presence of phospholipase A₂ (Vipera russelli) was compared to that from liposomes comprised of phospholipids. Phospholipase A₂ more readily attacked myelin and synaptic membranes than liposomes prepared from total phospholipids derived from myelin. Although hydrolysis of liposomal phospholipids occurred in the absence of added calcium, the presence of 2mm CaCl₂ or 2% bovine serum albumin significantly enhanced the phospholipase attack of liposomes, but not synaptic membranes or myelin. Phospholipase exhibited a marked preference for phospholipids containing docosahexaenoic acid (226) in the synaptic membranes, while with liposomes the pattern of released fatty acid reflected the fatty acid composition in the two-position of the phospholipids. Although either calcium or albumin markedly increased the phospholipase hydrolysis of liposomes, neither affected the hydrolysis of synaptic membranes or the pattern of fatty acid release from liposomes. It was concluded that the nonlipid constituents, particularly the proteins, of biomembranes were responsible for the organization of the phospholipids and accounted for the observed differences between liposomes and synaptic membranes with respect to enzymic accessibility.     

Lysophosphatidylcholine sensitizes lipid extracts of pulmonary surfactant to inhibition by serum proteins

Interactions between serum protein and lysophospholipid inhibitors of pulmonary surfactant were examined in vitro using a pulsating bubble surfactometer. In previous studies a particular batch of Lipid Extract Surfactant (LES) was observed to be unusually sensitive to inhibition by fibrinogen. This sample was found to contain an abnormally high concentration of lysophosphatidylcholine (lysoPC). Addition of exogenous lysophospholipid to LES at similar concentrations sensitized the surfactant to inhibition by fibrinogen. Sensitization to inhibition by lysoPC is also observed with fetal bovine serum. Under the conditions used, inhibition by bovine serum albumin was not affected. Whereas only small amounts of lysoPC (1 mol% added) maximally sensitize LES to inhibition by fibrinogen, co-addition of equal amounts of palmitic acid can partially offset this effect at low lysoPC concentrations (less than 5 mol%). Lipid Extract Surfactant was digested with phospholipase A2 to mimic the generation of endogenous lysoPC at the expense of surfactant lipids. Digestion of 2-3% of the phosphatidylcholine to lysophosphatidylcholine vastly sensitized the surfactant to inhibition by fibrinogen. These results suggest that the degradation of surfactant phospholipids by phospholipase A2 to lysophospholipids could contribute to the development and progression of adult and neonatal respiratory distress syndromes.     

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.     

Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A₂ (PLA₂)-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resiembedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA₂, which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mincralization suggests some hypotheses on the possible involvement of these molecules in the mineralphase deposition process.     

Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures

We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.     

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.     

Lipid composition of Trypanosoma cruzi.

1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35% of phospholipids and 65% of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4%), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32P showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.     

Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by other unsaturated lipids.

A study was undertaken on the possible involvement of phospholipids on stereospecific opiate binding to a rat brain membrane fraction comprised mainly of synaptic membranes. The addition of acidic phospholipids such as phosphatidylserine, phosphoinositides, and phosphatidic acid significantly enhanced opiate binding. With the exception of phosphatidylserine, when the acidic phospholipids contained a polyunsaturated acyl group, they were actually inhibitory, along with neutral phospholipids derived from brain. Both the C18:0, C18:1 form (derived from myelin) and the C18:0, C22:6 form of phosphatidylserine (derived from synaptic membranes) produced as much as a 45% enhancement in opiate binding. Unsaturated fatty acids were highly inhibitory, the degree of inhibition being related to the degree of unsaturation. Both phospholipase A and C were inhibitory; and the inhibitory effect of A could not be prevented by albumin or overcome with the addition of phosphatidylserine. With the use of the cross-linking agent, dinitrodifluorobenzene, it could be demonstrated that the phosphatidylserine of synaptic membranes appeared to be preferentially associated with membrane protein. The enhancement of opiate binding by phosphatidylserine diminished with increasing degree of cross-linking.     

Evidence for and subcellular localization of a ca-stimulated phospholipase d from maize roots

Autolytic lipid changes in corn (Zea mays L.) root crude homogenates and isolated membranes were examined by the use of high performance thin-layer chromatography. In the absence of added CaCl₂, losses in phosphatidylcholine and other phospholipids corresponds to increase in fatty acids without the accumulation of either phosphatidic acid or lyso-phosphatidylcholine. However, in the presence of 1 millimolar CaCl₂, phosphatidylcholine concentrations declined more rapidly with an immediate increase in phoshatidic acid, and slower rate of fatty acid accumulation. Autolytic phospholipid degradation yielded primarily free fatty acids in the absence of Ca and phosphatidic acid in the presence of 1 millimolar CaCl₂, suggesting the presence of an acyl hydrolase and phospholipase D activities. Differential centrifugation studies indicate that 50 to 80% of the crude homogenate's phospholipase D activity is membrane-bound. Density centrifugation experiments suggest that the membrane-bound phospholipase D activity is localized primarily on mitochondrial membranes.     

The stimulation by transmitter substances and putative transmitter substances of the net activity of phospholipase A2 of synaptic membranes of cortex of guinea-pig brain.

1. The distribution of the hydrolyses of phosphatidylcholine by phospholipase A2 and phospholipase A1, and the hydrolysis of lysophosphatidylcholine by lysophospholipase, in subcellular and subsynaptosomal fractions of cerebral cortices of guinea-pig brain, was determined. 2. Noradrenaline stimulated hydrolysis by phospholipase A2 in whole synaptosomes, synaptic membranes and fractions containing synaptic vesicles. 3. Stimulation of hydrolysis by phospholipase A2 in synaptic membranes by noradrenaline was enhanced by CaCl2, and by a mixture of ATP and MgCl2. The optimum concentration of CaCl2, in the presence of ATP and MgCl2, for stimulation by 10 muM-noradrenaline was in the range 1-10muM. The optimum concentration for ATP-2MgCl2 in the presence of 1 muM-CaCl2 was in the range 0.1-1mM. 4. Hydrolysis by phospholipase A2 of synaptic membranes was also stimulated by acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine), histamine, psi-aminobutyric acid, glutamic acid and aspartic acid. With appropriate concentrations of cofactors, sigmoidal dose-response curves were obtained, half-maximum stimulations being obtained with concentrations of stimulant in the range 0.1-1muM. 5. Taurine also stimulated hydrolysis of phosphatidylcholine by phospholipase A2. There were only slight stimulations with methylamine, ethylenediamine or spermidine. No stimulation was obtained with glucagon.     

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A2 of varying pI values. The susceptibility of platelet phospholipids to basic phospholipase A2 of Naja nigricollis (pI 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244-258). The susceptibilities of platelet phospholipids to acidic phospholipase A2 of Naja naja atra (pI 5.2) and to neutral phospholipase A2 of Hemachatus haemachatus (pI 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: the PC and the PE domains exist on the platelet membrane surface; and the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.     

Phospholipid synthesis by extracellular phospholipase A2 in organic solvents

The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical environment.