Neutral Phospholipase Research Articles

In silico Validation of Anti-Russell’s Viper Venom Activity in Phyllanthus emblica L. and Tamarindus indica L.

To validate the efficacy of viper anti-venom activity, a total of 79 phytochemicals from Phyllanthus emblica and 59 phytochemicals from Tamarindus indica were docked against each of the nine selected viper venom proteins such as basic phospholipase A2 VRV-PL-VIIIa, anticoagulant class II phospholipase A2, acidic phospholipase A2 Drk-a1, neutral phospholipase A2 RVV-PFIIc, L-amino acid oxidase, Russell's viper venom serine proteinase, RVV-V, cytotoxin drCT-1, dabocetin alpha subunit and cysteine-rich secretory protein Dr-CRPK using the tool Autodock. The structure of three proteins viz basic phospholipase A2 VRV-PL-VIIIa (PDB id-1OXL), anticoagulant class II phospholipase A2 (PDB id- 1VIP) and Russell's viper venom serine proteinase RVV-V (PDB id- 3S9A) were downloaded from RCSB Protein Data Bank and remaining targets 3D structures except neutral phospholipase A2 RVV-PFIIc were modelled using the tool SWISS-MODEL. The templates for modelling the structures were selected through BLASTp analysis. The structure of the neutral PLA2 was modelled using the tool I-TASSER. The stability of all the modelled structures were confirmed by Ramachandran Plot and the active site were detected using the tool MetaPocket2. The docked results revealed that both plants contain several inhibitory molecules against all the target proteins and certain compounds such as stigmasterol, β-sitosterol and campesterol, were present in both plants and each of these molecule can inhibit all venom proteins. The overall results substantiate the traditional use of these plants as antidote to snake bites. However, in vitro and in vivo experimental demonstration is essential for practical application of forgoing findings.

Mechanism of in vivo anticoagulant and haemolytic activity by a neutral phospholipase A2 purified from Daboia russelii russelii venom: Correlation with clinical manifestations in Russell's Viper envenomed patients

A 13.0 kDa neutral phospholipase A2 (NEUPHOLIPASE) purified from venom of Daboia russelii russelii from eastern India was identified by peptide mass fingerprinting analysis. It exerted dose-dependent PLA2, anticoagulant and indirect haemolytic activities. NEUPHOLIPASE showed preferential binding followed by hydrolysis of phosphatidylserine > phosphatidylcholine >> phosphatidylethanolamine. Circular dichroism analysis of NEUPHOLIPASE showed a high content of alpha helix (54.6%) followed by beta-turn (29.7%) in its secondary structure. Gas-chromatographic analysis of plasma from PLA2-treated mice suggested preferential hydrolysis of pro-coagulant phospholipid PS was the primary mechanism to account for in vivo anticoagulant effect of NEUPHOLIPASE. The NEUPHOLIPASE-treated mice blood showed a significant decrease (p < 0.01) in WBC as well as RBC counts with a corresponding decline in Hb content due to indirect damage to erythrocyte membranes by plasma phospholipids hydrolysis products rather than the direct haemolytic activity of PLA2 under study. NEUPHOLIPASE was non-lethal to BALB/c mice, however; it was detrimental to liver of treated-mice. Pathological symptoms observed in NEUPHOLIPASE-treated mice were correlated with the actual clinical manifestations in Russell's Viper envenomed patients from eastern India indicating some contribution of NEUPHOLIPASE in Russell's Viper venom induced toxicity and pathogenesis.

A secretory phospholipase A2-mediated neuroprotection and anti-apoptosis

BackgroundPhospholipase A2 liberates free fatty acids and lysophospholipids upon hydrolysis of phospholipids and these products are often associated with detrimental effects such as inflammation and cerebral ischemia. The neuroprotective effect of neutral phospholipase from snake venom has been investigated.ResultsA neutral anticoagulant secretory phospholipase A2 (nPLA) from the venom of Naja sputatrix (Malayan spitting cobra) has been found to reduce infarct volume in rats subjected to focal transient cerebral ischemia and to alleviate the neuronal damage in organotypic hippocampal slices subjected to oxygen-glucose deprivation (OGD). Real-time PCR based gene expression analysis showed that anti-apoptotic and pro-survival genes have been up-regulated in both in vivo and in vitro models. Staurosporine or OGD mediated apoptotic cell death in astrocytoma cells has also been found to be reduced by nPLA with a corresponding reduction in caspase 3 activity.ConclusionWe have found that a secretory phospholipase (nPLA) purified from snake venom could reduce infarct volume in rodent stroke model. nPLA, has also been found to reduce neuronal cell death, apoptosis and promote cell survival in vitro ischemic conditions. In all conditions, the protective effects could be seen at sub-lethal concentrations of the protein.

Enzymatic characterization of phospholipase D of protozoan Tetrahymena cells.

Phospholipase D (PLD), which is present in plant, bacterial, and mammalian cells, has been proposed to be involved in a number of cellular processes including transmembrane signaling and membrane deterioration. We demonstrated the existence of evolutionally related PLD activity in the unicellular eukaryotic protozoan Tetrahymena. The partial characterization of this enzyme showed that PLD in Tetrahymena cells was a neutral phospholipase, which catalyzed both transphosphatidylation and hydrolysis reac tions. The activity was markedly stimulated by phosphatidylinositol 4, 5-bisphosphate (PIP2) but was insensitive to phorbol 12-myristate 13-acetate (PMA) and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), suggesting that it is a PIP2-dependent PLD and that protein kinase C (PKC) and GTP-binding proteins are not implicated in the regulation of this enzyme. For its maximal activity Ca2+ was not required. This enzyme was also capable of hydrolyzing phosphatidylcholine (PC) but not phosphatidylethanolamine (PE), implying that PC was a preferred substrate. Subcellular fractionation showed that PLD-like activity localized mainly to the membrane fraction, especially microsomes. As an initial step to explore the functions of PLD in Tetrahymena, the PLD-like activity was determined during the different culture phases, and it was found to be significantly and transiently elevated in the early logarithmic phase, indicating its possible role in the development of Tetrahymena.

Effect of rate of pH decline on muscle enzyme activities in two pig lines

Muscle enzyme activities were measured in m. transversus abdominis (TA) samples of two pig lines, differing in stress sensitivity and in breeding objectives. Animals of line one were stress sensitive and selected for a high carcass lean content (LEAN), those of line two were homozygous stress resistant and selected for maximum live weight gain (GROWTH). Muscle samples of 16 pigs per line were excised at 1 h and sampled at 3 h post mortem. Boars, gilts and barrows were almost equally represented. Pigs of line GROWTH had a higher fat and lean tissue gain ( P<0.01) and a lower carcass lean content ( P<0.001) than those of line LEAN. Lower activities of μ-calpain, m-calpain, pyroglutamyl aminopeptidase I, acid lipase and neutral phospholipase were measured in TA muscle samples of line LEAN compared with line GROWTH ( P<0.05). Activities of dipeptidyl peptidase IV and acid phospholipase tended to be somewhat lower for line LEAN, whereas activities of calpastatin, cathepsin (B+L) and cathepsin D were not different. Sex-class effects were analogous to line effects. Significant correlation coefficients between activities of several enzymes and pH values 1 h post mortem, both within and across lines, suggest that lower enzyme activities are a consequence of a more pronounced denaturation due to a faster post mortem pH decline.

The determination of lipase and phospholipase activities in gut contents of turbot (Scophthalmus maximus) by fluorescence-based assays

To study the potential of fluorescence based assays in the study of lipid digestion in fish, acyl esters of 4-methylumbelliferone and 1-acyl-2-[6 (7 nitro-1,3 benzoxadiazol-4-yl)amino]caproyl labeled phosphatidylcholine compounds (NBD-PC) were used as substrates for the assay of neutral lipase and phospholipase, respectively, in the gut contents of turbot. 4-Methylumbelliferyl hepatanoate (4-MUH) was hydrolysed at a higher rate than the butyrate or oleate esters whilst the hexanoic (C6) ester of NBD-PC was a more convenient substrate for the phospholipase assay than the dodecanoic (C12) ester. Neutral lipase activity was almost 10% higher when 50 mm potassium phosphate buffer pH 7.8 was used instead of 0.01 m citrate/sodium phosphate buffer pH 7.2. Both assays were very sensitive: neutral lipase and phospholipase activities were detectable at a minimum protein concentration in the digesta of 0.04 and 1.25 mg/ml, respectively. When the variations in lipolytic activities with gut segment and with size of fish were examined neutral lipase activity was always found to be higher in the hindgut and rectum segments than in the foregut. Although phospholipase activity was also found to be highest in the hindgut of the largest fish examined (av. wt. 182.3g), in fish of average weight 8g fish the activity was similar in all three segments. In the digesta from the whole gut of smaller fish (av. wt. 0.2, 0.6 and 1.43g) neutral lipase and phospholipase activities increased with increasing body mass when expressed as per ml of digesta. It is concluded that fluorescence-based assays are applicable to the study of lipid digestion in fish of different size.

Non-crystallographic symmetry of crystal of neutral phospholipase A2 fromAgkistrodon halys Pallas

Agkistrodotoxin, a neutral phospholipase A(2) with high presynaptic neurotoxicity from the venom ofAgkistrodon halys Pallas, has been crystallized by hanging drop vapor diffusion method. The crystal belongs to P2(1) space group with the cell dimensionsa = 10.836 nm,b = 8.486nm,c = 7.082nm, beta = 109.87 degrees , showing C2 pseudo-symmetry. Diffraction data to 0.26 nm resolution have been collected on a Siemens X-200B area detector. C2 pseudo symmetry suggests that there exists a non-crystallographic two-fold axis parallel to crystallographicb axis. Selfrotation function calculation with different integrated radius and resolution ranges using the program POLARRFN yields four stable high peaks corresponding to three more non-crystallographic two-fold axis and one special non-crystallographic symmetry. The molecules in the asymmetric unit are suggested to be arranged in a manner ofdimer of dimers" by inference.

Characterisation of a purified phospholipase A 2 from the venom of the Papuan black snake ( Pseudechis papuanus)

A neutral phospholipase A 2 (PLA 2) was separated from Pseudechis papuanus venom by a two-stage FPLC procedure of cation exchange and phenyl-Superose chromatography. It had a molecular mass of 15 kDa and a lower LD 50 value than a co-separated haemorrhagic fraction, indicating a higher lethal potency. In vitro tests confirmed the powerful inhibition of platelet aggregation by the PLA 2 and strong anticoagulant activity initially observed with whole venom. Ultrastructural studies showed that platelets lost their discoid shape and developed membranous projections with a general decrease in electron-density of the cytosol and disruption of the microfilaments following incubation with the enzyme. Amino acid sequence analysis of the N-terminus and some internal peptides demonstrated a high degree of homology with PLA 2s from other Pseudechis venoms. Our results indicate that this fraction is the main agent responsible for the haemostatic disorders in envenomed patients.

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.

Subcellular distribution of fatty acids, phospholipids and phospholipase A 2 in human neutrophils

We have examined the putative relationship between the presence of neutral pH optimum phospholipase A 2 activities and the phospholipid and fatty acid composition in the distinct fractions obtained after subcellular fractionation in a continuous sucrose gradient of human neutrophils. Neutral phospholipase A 2 activity was found mainly in the plasma membrane, the azurophilic granules and an unidentified fraction which was a little denser than that containing specific granules and endoplasmic reticulum. These differently located phospholipase A 2 activities resulted in activation upon calcium ionophore A23187-cell treatment, but the phospholipase A 2 activity found in the unidentified region showed the highest degree of activation after cell treatment with A23187 and hydrolyzed preferentially ethanolamine-containing glycerophospholipids. The subcellular distribution of inositol-containing phosphoglycerides was bimodal, with a main peak at the plasma membrane and another peak at the azurophilic granule region. Choline- and ethanolamine-containing glycerophospholipids showed a broad distribution throughout the gradient, with preponderance in the denser part of the gradient, where the intracellular organelle phospholipase A 2 activities were located. Interestingly, ethanolamine-containing glycerophospholipids were shown to be enriched in fractions containing the phospholipase A 2 which hydrolyzes preferentially this phospholipid class. Most of the arachidonic acid present in the postnuclear fraction of human neutrophils was found in the plasma membrane and resulted to be the only fatty acid released during A23187 stimulation. Thus, plasma membrane phospholipase A 2 seems to be involved mainly in the release of arachidonic acid in A23187-stimulated cells. These findings indicate that differences in substrate specificity found in vitro among the phospholipase A 2 activities located in distinct subcellular particulates might be a reflection of the distinct phospholipid and fatty acid composition in these particulates.

A neutral phospholipase D activity from rat brain synaptic plasma membranes. Identification and partial characterization.

Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.

Biochemical characterization of phospholipase D activity from human neutrophils

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol-1-phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2(+)-independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350-400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was detected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal-transducing processes during cell stimulation in human phagocytes.

Amino acid sequence of a neutral phospholipase A2 (III) in the venom of Bungarus fasciatus.

Two phospholipases were found in the venom of Bungarus fasciatus, one in fraction III, the other in fraction X of the chromatographic separation. A neutral PLA2(III) purified from fraction III was subjected to amino acid sequencing by means of an automated sequenator applied to the intact RCM-PLA2 (III) and the individual peptides obtained from HPLC separation of the three types of enzymatic peptides. PLA(III) was shown to consist of 118 amino acid residues with 14 half-cystines. It is 65% homologous to the basic PLA2 obtained from fraction X.

A novel phospholipase A 2 associated with nuclear matrix: stimulation of the activity and modulation of the Ca 2+ dependency by polyphosphoinositides

Neutral phospholipase A 2 activity, which hydrolyzed phosphatidylcholine and phosphatidylethanolamine with the same efficiency, was identified in the nuclear matrix prepared from purified nuclei of rat ascites hepatoma cells (AH 7974). The enzyme activity was optimal at pH 7.0 and required Ca 2+ absolutely. Concentrations of Ca 2+ for a maximal and a half-maximal activation were 1·10 −2 and 1·10 −3 M, respectively, and little activity was detected at Ca 2+ concentrations lower than 1·10 −5 M. Addition of acidic phospholipids markedly stimulated the enzyme activity, and further, lowered the minimum Ca 2+ concentration required for activation. In particular, the polyphosphoinositides phosphatidylinositol 4-monophosphate and 4,5-diphosphate were most effective. These two polyphosphoinositides lowered the Ca 2+ concentration required for half-maximal activation to 10 −5 M and dramatically stimulated the activity at that Ca 2+ concentration (> 30-fold). The neutral phospholipase A 2 activity such as characterized in the present study was very low in the other subcellular fractions including mitochondria, microsome, plasma membrane and cytosol.

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.

Propranolol inhibition of the neutral phospholipases a of rat heart mitochondria, sarcoplasmic reticulum and cytosol

Membrane damage caused by phospholipase A action is thought to be an important factor in ischemic myocardial injury. Propranolol has been shown previously to have beneficial effects in both animal experiments and clinical trials, and it has membrane-stabilizing properties in vitro. To investigate the possibility that these effects might be due, in part, to effects on phospholipases, we determined the effects of propranolol on rat heart phospholipases A at physiological pH using small unilamellar liposomes of di[1- 14C]oleoylphosphatidylcholine as substrate. Propranolol inhibited heart phospholipases A in vitro.The concentration required to give 50% inhibition was 0.2 mM for the mitochondrial and cytosolic phospholipases A and 2.9 mM for sarcoplasmic reticulum phospholipase A. The binding of [4- 3H]propranolol to fresh membrane preparations was studied using an ultracentrifugation method. Propranolol bound readily to both membrane fractions in vitro with no significant difference in the saturation number (0.20 to 0.28 mol drug per mol phospholipid) but the association constant, K A, was higher for mitochondrial membranes (3760 ± 350) than for the sarcoplasmic reticulum (2190 ± 390). Our results show that propranolol inhibited heart phospholipases A in vitro at physiological pH. The mitochondrial and cytosolic phospholipases A were more susceptible to inhibition than the phospholipase A of sarcoplasmic reticulum. Propranolol bound to mitochondria and sarcoplasmic reticulum in vitro, suggesting the possibility that propranolol binding to heart membranes in vivo could result in drug concentrations in these membranes high enough to inhibit phospholipase A. This could represent an additional mechanism by which propranolol exerts beneficial effects in myocardial ischemia

Effects of chronic amiodarone treatment on cat myocardial phospholipid content and on in vitro phospholipid catabolism.

Amiodarone is used extensively for the chronic treatment of life-threatening arrhythmias caused by ischemic heart disease. However, chronic therapy with this agent results in phospholipidosis in various tissues and it has been suggested that the inhibition of lysosomal phospholipase A by this drug contributes to this abnormality. Exogenous amiodarone has been shown to inhibit purified rat liver lysosomal phospholipase A1, as well as acid phospholipase activities of alveolar macrophage homogenates and those of snake venom phospholipase A2 and bacterial phospholipase C. The effects of drug treatment on heart have not been explored. The results described here demonstrate that amiodarone also significantly increases (37%, p less than 0.001) phospholipid content in cat hearts. This increase is proportionately distributed to all major phospholipid classes, with the exception of sphingomyelin which appears to increase more than the others. In addition, the data also show that following amiodarone treatment, the endogenous drug levels in the heart were sufficient to reduce in vitro losses of membrane phospholipid at 37 degrees C by inhibiting a variety of endogenous phospholipases at physiological (7.4), ischemic (6.2) and acidic (5.0) pH values. This protection is more pronounced at acidic pH values than at physiological pH. Endogenous amiodarone also affects myocardial phospholipase activities towards exogenous phosphatidylcholine and again the extent of inhibition is more at acidic pH. These results suggest that amiodarone induces phospholipidosis in the heart by inhibiting phospholipid catabolism and that its antiarrhythmic properties may reside in its ability to modulate alkaline, neutral and acid phospholipase activities in ischemia. To what extent amiodarone metabolites (desethylamiodarone and bis-desethylamiodarone) are involved in these actions remains to be determined.

Effect of dietary lipids on the lipid composition and phospholipid deacylating enzyme activities of rat heart.

Rats were fed lard-enriched (17%) or corn oil-enriched (17%) diets and were compared with rats fed a low fat (4.5%) diet. Cardiac protein, DNA, phospholipid (PL) and fatty acid (FA) compositions were analyzed. Neutral phospholipase A, lysophospholipase and creatine kinase activities in the membrane and cytosolic compartments were also investigated. No significant modification of cardiac protein, DNA nor PL was observed among the three groups. Some alterations appeared in the FA composition. A lard-enriched diet induced a significant increase of 22:5n-3 and 22:6n-3 in heart phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas a linoleic acid-rich diet induced a specific increase of 22:4n-6 and 22:5n-6 in these two major PL. Compared to rats fed the low fat diet, membrane-associated phospholipase A activity, measured by endogenous hydrolysis of membrane PC and PE, showed a significant increase (+45%) for both PL in rats fed corn oil. However, the activity of membrane-associated phospholipases, measured with exogenous [1-14C]dioleoyl PC, was not different among the three groups of rats. Cytoplasmic activity was decreased in rats fed corn oil, and lysophospholipase and creatine phosphate kinase activities were not significantly affected by diet. FA modification of the long chain n-6 FA induced by corn oil may be responsible for the observed increase in phospholipase activity. Physiological implications are suggested in terms of membrane degradation and prostaglandin production.

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 A 2 of varying p I values. The susceptibility of platelet phospholipids to basic phospholipase A 2 of Naja nigricollis (p I 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244–258). The susceptibilities of platelet phospholipids to acidic phospholipase A 2 of Naja naja atra (p I 5.2) and to neutral phospholipase A 2 of Hemachatus haemachatus (p I 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A 2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A 2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A 2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A 2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A 2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A 2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A 2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: (1) the PC and the PE domains exist on the platelet membrane surface; and (2) the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.

Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas

Y.-C. Chen, J. M. Maraganore, I. Reardon and R. L. Heinrikson. Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas. Toxicon 25, 401 – 409. 1987.—Three monomeric phospholipases A 2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A 2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air — water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A 2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A 2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic enzyme exhibits the abilities to hemolyze erythrocytes and to hydrolyze phospholipids of E. coli membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.