Hemolytic assay for venom phospholipase A2

A rapid and sensitive spectrophotometric assay for venom phospholipase A₂ based on the hemolysis of guinea pig erythrocytes in the presence of decomplemented serum and cardiotoxin (direct lytic factor) is described. This assay is particularly useful for rapid multisample analyses, such as those used in monitoring chromatography fractions, and is specific for phospholipase A₂ in she presence of other potentially hemolytic venom components. The hemolytic mechanism is shown to be a combination of the action of lysophospholipids liberated from lipoproteins in the serum and the synergistic action of phospholipase A₂ and cardiotoxin on the erythrocyte membrane.     

Action of phospholipase A2 and phospholipase C on Escherichia coli

The action of exogenous phospholipases on Escherichia coli has been examined. Cells harvested in late log phase were found to be completely resistant to the action of phospholipases A₂ and C. Treatment of cells with Tris and EDTA was required to make the phospholipids in the cell accessible to these phospholipases. Phospholipase A₂ hydrolyzed mainly phosphatidylethanolamine and phosphatidylglycerol, whereas phospholipase C preferentially degraded phosphatidylethanolamine.During the EDTA treatment, an endogenous phospholipase A₁ or a lysophospholipase (or both) was unmasked which caused the formation of free fatty acids in experiments in which no phospholipase was added and which degraded some of the lysophospholipids formed by phospholipase A₂.The cells were rapidly killed by the successive Tris-EDTA-phospholipase treatment, but no cell disintegration was observed.     

Evidence of phospholipase A2 in the sea urchin egg: Its possible involvement in the cortical granule reaction

Fertilization of the sea urchin egg involves an exocytotic event known as the cortical granule reaction (CGR). In many cell systems, phospholipase A₂ is implicated in regulation of the secretory event. Indirect evidence suggests that phospholipase A₂ mediates the CGR; however, there has been no direct demonstration of phospholipase A₂ activity in the sea urchin egg. We report here evidence of phospholipase A₂ activity in egg homogenate of the sea urchin Lytechinus pictus. The enzyme was calcium-dependent and had a pH optimum near the intracellular pH of the unfertilized egg. Neither exogenous calmodulin nor trifluoperazine had any apparent effect on enzyme activity. Quinacrine, a phospholipase A₂ inhibitor, blocked the enzyme activity in the egg homogenate. In intact eggs, quinacrine blocked the CGR in a dose-dependent, egg-concentration-dependent manner. The inhibitory effect of quinacrine on the CGR could not be overcome by the phospholipase A₂ activator melittin or by the calcium ionophore A23187. Quinacrine did not inhibit sperm-egg binding or sperm incorporation. These results lend further support to the hypothesis that phospholipase A₂ is involved in the CGR.     

Phosphatidylcholine metabolism in human endothelial cells: modulation by phosphocholine

Phosphatidylcholine is the principal phospholipid in mammalian tissues, and a major source for the production of arachidonic acid. In this study, the effect of exogenous phosphocholine, a precursor of phosphatidylcholine biosynthesis, on the metabolism of phosphatidylcholine in human umbilical vein endothelial cells was investigated. Incubation of endothelial cells with exogenous phosphocholine at concentrations of 1 to 5 mM was found to inhibit choline uptake and its subsequent incorporation into phosphatidylcholine. Phosphocholine appeared to inhibit choline uptake in a competitive manner. Since phosphatidylcholine is metabolized mainly by the action of phospholipase A₂, with the release of arachidonic acid and other fatty acids, the effect of phosphocholine on arachidonic acid release in endothelial cells was also examined. The induction of arachidonic acid release by ATP was enhanced in cells treated with 1 mM phosphocholine. In vitro assays of phospholipase A₂ activity in cells incubated with phosphocholine, however, did not produced any significant change in the activity of this enzyme. The results of this study show that phosphocholine modulates the biosynthesis and catabolism of phosphatidylcholine in an indirect manner.     

Pharmacokinetics of Naja sumatrana (Equatorial Spitting Cobra) Venom and Its Major Toxins in Experimentally Envenomed Rabbits

Background

The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom''s composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A₂, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits.

Principal findings

The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A₂ and whole venom (T_max = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (F_i.m. = 81.5%) compared to that of the phospholipase A₂ (F_i.m. = 68.6%) or cardiotoxin (F_i.m. = 45.6%). The incomplete absorption of the phospholipase A₂ and cardiotoxin may infer the toxins'' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions.

Conclusion/Significance

Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.     

Phospholipid,an enhancing component in the synergistic factor of a granulosis virus of the armyworm,Pseudaletia unipuncta

The synergistic factor (SF) in the capsule of a granulosis virus (Hawaiian strain) of the armyworm, Pseudaletia unipuncta, contained polypeptides and phospholipids. Its molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 126,000 ± 8,700. The capsule proteins were digested by a proteinase released from the capsule under alkaline conditions, and by trypsin added to the proteinase-free capsules. Neither enzyme affected the synergistic factor or its activity. The synergistic factor was slowly depolymerized by 2% sodium dodecyl sulfate and was more rapidly depolymerized when phospholipase C (phosphatidylcholine cholinephosphohydrolase) was also added. Phospholipase C alone did not decompose the synergistic factor, but it did destroy the capacity of the synergistic factor to enhance the nuclear polyhedrosis virus. In contrast, phospholipase A₂ (phosphatidyl 2-acylhydrolase) had no effect on the synergistic factor. The different reactions of the two phospholipases on the synergistic factor suggested that the hydrophilic group of the phospholipid was exposed to the action of phospholipase C and was associated with the synergistic activity. This interpretation was supported by the detection of a phospholipid in the SF by thin-layer chromatography.     

Ceramides perturb the structure of phosphatidylcholine bilayers and modulate the activity of phospholipase A2

The effects of a series of ceramide analogs with acyl chain lengths of 2, 6, 8 and 16 on the structure of dipalmitoylphosphatidylcholine (DPPC) bilayers and cobra venom phospholipase A₂ (PL-A₂) activity were studied using ²H-NMR and specific enzymatic assays. C₂-ceramide did not induce a significant effect on the structure of DPPC bilayers and did not alter PL-A₂ activity. C₆- and C₈-ceramides increased the ordering of the DPPC acyl chains, correlating with the inhibition of PL-A₂ activity which was probably due to the increased lateral surface pressure. The long-chain C₁₆-ceramide induced lateral phase separation of the bilayers into gel and liquid crystalline domains and activated PL-A₂, as does natural ceramide (Huang et al. 1996). Taken together, the results strongly suggest a correlation between membrane defects induced by ceramide analogs and their effects on phospholipase A₂ activity. Furthermore, the effects of short-chain ceramides on PL-A₂ are different from those of natural ceramide, indicating that the cell-permeable short-chain ceramide analogs, widely used to study the sphingomyelin-dependent cellular signal transduction pathway, may not completely mimic the natural product. Received: 8 July 1997 / Accepted: 19 January 1998     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)₂D₃ and 1,25-(OH)₂D₃ regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)₂D₃ affecting primarily growth zone (GC) cells and 24,25-(OH)₂D₃ affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)₂D₃ has been shown to increase phospholipase A₂ activity in GC, while 24,25-(OH)₂D₃ has been shown to decrease phospholipase A₂ activity in RC. Stimulation of phospholipase A₂ in GC caused an increase in PKC, whereas inhibition of phospholipase A₂ activity in RC cultures increased both basal and 24,25-(OH)₂D₃-induced PKC activity, suggesting that phospholipase A₂ may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A₂ inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A₂ activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A₂ inhibitors decreased both basal and 1,25-(OH)₂D₃-induced PKC activity in GC. When phospholipase A₂ activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)₂D₃-induced PKC activity in RC and increased 1,25-(OH)₂D₃-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A₂ activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A₂ activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A₂. These effects are cell maturation- and time-dependent and metabolite-specific. J. Cell. Physiol. 176:516–524, 1998. © 1998 Wiley-Liss, Inc.     

Possible involvement of phospholipase A2 and cyclooxygenase in stimulatory action of L-histidine on protein synthesis in L6 myotubes

Effects of L-histidine and related compounds on protein synthesiswere studied in cultured L6 myotubes. L-Histidine specifically stimulated protein synthesis, whereas D-histidine, histamine, L-arginine and L-lysine did not. Inhibitors of phospholipase A₂, phospholipase C and cyclooxygenase intercepted the stimulatory action of L-histidine on protein synthesis, while inhibitors of protein kinase C and 5-lipoxygenase did not. These results suggest an involvement of phospholipase A₂ and cyclooxygenase in the stimulatory action of L-histidine on protein synthesis in L6 myotubes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and preliminary characterisation of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix)

Two toxic phospholipases A have been isolated from the venom of the Malayan cobra (Naja naja sputatrix). The phospholipases A were purified by successive ion-change chromatography on SP-Sephadex C-25, Sephadex G-75 gel filtration chromatography and successive Bio-Rex 70 ion-exchange chromatography. The purified toxic phospholipases A were homogeneous electrophoretically. They were designated as sputatrix phospholipase A-I and sputatrix phospholipase A-II. Positional specificity studies showed that they belong to the A₂-type phospholipase A. The medium lethal dose 50% (LD₅₀) values of the two phospholipases A are 0.27 and 0.28 μg/g, respectively, by intravenous injection and 1.05 and 1.00 μg./g, respectively, by intraperitoneal injection. The molecular weights of the two enzymes are 14 000 as determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Amino acid composition of sputatrix phospholipase A-I differs from sputatrix phospholipase A-II only by having one extra amino acid: a glutamic acid. Amino acid compositions of the two enzymes are also similar to those of other cobra venom phospholipases A.     

Inhibition of phospholipase A2 activity by fluosol®, an artificial blood substitute

This paper described the effect of Fluosol® on phospholipase A₂ activity both _vitro and in vivo. This compound inhibited both postheparin plasma and hepatic phospholipase A₂ activities. Furthermore, in Fluosol transfussed rats the postheparin plasma phospholipase A₂ activity was 41% below that observed in control animals. The inhibition of phospholipase A₂ activity was due to the micellar forms of pluoronic, F-68, a detergent present in Fluosol. It is postulated that the inhibition of phospholipase A₂ activity by Fluosol might interfere with the release of arachidonic acid, a substrate used in biosynthesis of prostaglandin.     

Phospholipase A2 Down-Regulates the Affinity of [3H]AMPA Binding to Rat Cortical Membranes

Abstract: The effects of exogenous phospholipase A₂ on the binding of α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) to rat cortical membranes in the presence of the chaotrope potassium thiocyanate were assessed. Pretreatment of membranes with secretory phospholipase A₂ (sPLA₂) elicited a concentration-dependent decrease in specific [³H]AMPA binding due mainly to a decrease in affinity (K_D). This observation, together with protease inhibitor and western blot evidence, suggest that the sPLA₂ effect is not due to proteolysis. The sPLA₂-evoked decrease was temperature and calcium dependent. Inclusion of the specific inhibitor oleoyloxyethyl phosphocholine or preincubation of the enzyme with reducing agents to degrade its secondary structure significantly reduced the sPLA₂ inhibition. These results suggest that the effects of sPLA₂ arise from an enzymatic action rather than a competitive interaction at the AMPA binding site. However, arachidonic acid, a major metabolite of sPLA₂ action, did not cause a similar decrease in the affinity of [³H]AMPA binding. In contrast to the effects on [³H]AMPA binding, sPLA₂ caused an increase in [³H]CNQX binding, which is in accordance with the functionality of the AMPA receptor complex. These results suggest that sPLA₂ may play a role in the physiological and pathophysiological regulation of AMPA receptors.     

Study of histamine binding to h2-receptors of gastric mucosa membranes of the frogRana ridibunda

The stability was studied of histamine H₂-receptors and of histamine-sensitive adenylyl cyclase of the crude membrane fraction of the gastric mucosa of the frogRana ridibunda to the action of exogenous hydrolases, lipase (phospholipase C), protease (papain), glycosidase (sialidase), and blockers of free SH-groups (iodoacetate and N-ethylmaleimide). The action of these agents on the free and histamine-occupied H₂ -receptors of the frog gastric mucosa was analyzed by the amount of the bound ligand. The histamine binding to receptor increased the receptor vulnerability to the effect of phospholipase C, papain, and SH-reagents. Study of the action of hydrolases on the basal and stimulated, histamine-sensitive adenylyl cyclase activity revealed that phospholipase C caused a decrease of the basal and all kinds of the stimulated activity of adenylyl cyclase, while papain and sialidase only prevented the histamine stimulation of the enzyme. The obtained data indicate changes of the surface exposure of functional groups during the specific ligand-receptor interaction.     

Study of histamine binding to h2-receptors of gastric mucosa membranes of the frogRana ridibunda

The stability was studied of histamine H₂-receptors and of histamine-sensitive adenylyl cyclase of the crude membrane fraction of the gastric mucosa of the frogRana ridibunda to the action of exogenous hydrolases, lipase (phospholipase C), protease (papain), glycosidase (sialidase), and blockers of free SH-groups (iodoacetate and N-ethylmaleimide). The action of these agents on the free and histamine-occupied H₂-receptors of the frog gastric mucosa was analyzed by the amount of the bound ligand. The histamine binding to receptor increased the receptor vulnerability to the effect of phospholipase C, papain, and SH-reagents. Study of the action of hydrolases on the basal and stimulated, histamine-sensitive adenylyl cyclase activity revealed that phospholipase C caused a decrease of the basal and all kinds of the stimulated activity of adenylyl cyclase, while papain and sialidase only prevented the histamine stimulation of the enzyme. The obtained data indicate changes of the surface exposure of functional groups during the specific ligand-receptor interaction.     

The effect of hexadecylphosphocholine on the degradation of mitochondrial phospholipids

Hexadecylphosphocholine (HePC) is known as antitumor agent but the mechanism has not yet been understood. In rat liver mitochondria its effect on phospholipid transformation has been studied by quantitative HPTLC and phosphorus determination. From the results it can be concluded that HePC influences the activities of phospholipase A₂, phospholipase C, phospholipase D, and lysophospholipase A. The phospholipid transformation as well as the influence of HePC are affected by exogenous calcium ions. In the presence of calcium HePC has been found to inhibit enzyme activities, whereas in the absence of exogenous calcium ions enzymatic phospholipid transformations are activated or inhibited depending on HePC concentrations.     

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A₂ activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from $\text{E.}$$\text{coli}$ membranes. Phospholipase A₂ activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A₂ activity which was assayable in islet homogenates. Glucose stimulated phospholipase A₂ in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A₂ activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A₂ activity measured in the presence of high calcium concentrations. Phospholipase A₂ activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A₂ activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.     

Effect of hormones and antihormones on phospholipase A2 activity in human endometrial stromal cells

Phospholipase A₂ activity was studied in isolated human endometrial predecidual cells, and in human endometrium collected from day 19–23 of the menstrual cycle, by performing a radiochemical assay. Phospholipase A₂ activity on day 20 was significantly higher than other days (P < 0.001), and the activity was found to gradually decrease after day 20 of the menstrual cycle. The effects of the hormones estradiol and progesterone, and antihormones tamoxifen and RU 486, were studied on the phospholipase A₂ activity in isolated predecidual stromal cells. Estradiol produced a significant stimulatory effect (P < 0.001) on phospholipase A₂ activity in predecidual cells, and this effect was antagonized by tamoxifen. The combination of estradiol and tamoxifen was significantly different from estradiol alone (P < 0.001), but not from tamoxifen alone. RU 486 alone significantly increased (P < 0.001) phospholipase A₂ activity in predecidual stromal cells. However, progesterone had no effect on phospholipase A₂ activity in predecidual stromal cells.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

Nonantibiotic Properties of Tetracyclines: Structural Basis for Inhibition of Secretory Phospholipase A2

Secretory phospholipase A₂ is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A₂, which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 Å resolution of the minoTc complex of phospholipase A₂ (PLA₂) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca²⁺-binding loop, preventing Ca²⁺ binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA₂ is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA₂ was determined by surface plasmon resonance, resulting in a dissociation constant K_d = 1.8 × 10^− 4 M.     

Phospholipase A<Subscript>2</Subscript> Inhibitors Modulate the Effect of Trifluoperazine on the Intracellular Ca<Superscript>2+</Superscript> Concentration in Macrophages

Using Fura-2AM microfluorimetry, it was shown for the first time that phospholipase A₂ inhibitors 4-bromophenacyl bromide and glucocorticosteroids prednisolone and dexamethasone attenuate Ca²⁺ responses induced by neuroleptic trifluoperazine in macrophages. The results suggest the involvement of phospholipase A₂ and arachidonic acid metabolism cascade in the effect of trifluoperazine on intracellular Ca²⁺ concentration in macrophages.