Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

Q.H. Gong, S.J. Wieland, J.E. Fletcher, G.E. Conner and M.S. Jiang. Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

Factors influencing the hemolysis of human erythrocytes by cardiotoxins from Naja naja kaouthia and Naja naja atra venoms and a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom

The effects of red blood cell age and incubation conditions (temperature, divalent cation type and concentration, pH and glucose) on hemolysis induced by cardiotoxin fractions from Naja naja atra and Naja naja kaouthia venoms, a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom and bee venom phospholipase A2 were examined. Hemolysis by the snake venom toxins was dependent on red blood cell age (aged more susceptible than fresh) and the temperature of incubation (37 degrees C greater than 20 degrees C). Divalent cations at 0.5-2.0 mM enhanced (Ca2+) or slightly decreased (Sr2+, Ba2+) hemolysis due to N. n. kaouthia and N. n. atra toxins, and greatly decreased (Ca2+, Sr2+, Ba2+) hemolysis by these toxins at higher concentrations (5-40 mM). For the B. fasciatus phospholipase A2, Ba2+ and Sr2+ could not fully support hemolysis in any concentration while both low (less than 0.5 mM) and high (greater than 40 mM) Ca2+ enhanced hemolysis. Bee venom phospholipase A2 only induced hemolysis (greater than 10% at greater than 40 mM) at high concentrations of Ca2+. Increasing the pH from 7.5 to 8.5 greatly increased the levels of hemolysis by the snake venom toxins and enzyme. Glucose (5.3 mM) increased hemolysis by the snake venom components at low concentrations of divalent cations (2 mM) and slightly decreased hemolysis at high concentrations (40 mM). Treatment with p-bromophenacyl bromide abolished phospholipase A2 activity of bee venom and B. fasciatus phospholipases, but did not affect hemolytic potency of N. n. kaouthia or B. fasciatus toxins. A similar mechanism, which is independent of phospholipase A2 activity, may be involved in hemolysis by the N. n. kaouthia and N. n. atra cardiotoxins. The B. fasciatus cardiotoxin-like phospholipase A2 appears to have two mechanisms of hemolysis; the first is similar to that of the two typical cardiotoxins and the second appears dependent on phospholipase A2 activity and is only evident at high Ca2+ concentrations.     

Ceruleotoxin: identification in the venom of Bungarus fasciatus, molecular properties and importance of phospholipase A2 activity for neurotoxicity

Ceruleotoxin is a potent neurotoxin which was originally purified from a batch of venom labelled Bungarus caeruleus, from the Pasteur Institute. Since NOBLE et al. have shown that this batch differs in its protein composition from that of B. caeruleus provided by Miami Serpentarium, we decided to clarify this point by comparing the composition of venoms from various Bungarus species of several origins. Although individual variations exist between samples of the same species, the venom from B. multicinctus, B. caeruleus and B. fasciatus possess characteristic protein compositions which allowed us to identify the batch used to purify ceruleotoxin as a B. fasciatus venom. We identified and purified ceruleotoxin from each of the five samples of B. fasciatus venoms tested. We failed to find this neurotoxin in either B. multicinctus or B. caeruleus venoms. Purified ceruleotoxin is a slightly basic protein with an isoelectric point of 7.4 which possesses a significant phospholipase A2 activity (200 mumoles lecithin hydrolyzed per min per mg) and a high lethal potency (i.v. LD50 in mice 0.03-0.07 mg/kg). It is composed of two identical subunits of 13,000 mol. wt. which resemble pancreas and snake venom phospholipases in their amino acid composition. Like crotoxin, ceruleotoxin irreversibly blocks the postsynaptic response of Torpedo and Electrophorus electroplaques to cholinergic agonists without preventing the binding of acetylcholine to its receptor. By hydrolyzing critical lipids of the postsynaptic membrane, it stabilizes the acetylcholine receptor - ionophore assembly in a desensitized state.     

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.     

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—II: Pharmacological properties in relationship to enzymatic activity

Despite a remarkable degree of homology in amino acid sequence, the neutral phospholipase A₂ from Hemachatus haemachatus venom is much less toxic than the basic phospholipase A₂ from Naja nigricollis venom, the i.v. ld₅₀ in mice for the two being, respectively, 8.6 and 0.63 mg/kg. Similarly following intraventricular injection into rats, the neutral phospholipase showed convulsant and lethal dose₅₀ values of about 7.5 and 15 μg per rat, respectively, whereas corresponding values for the basic phospholipase were 0.5 and 0.5 μg per rat. Death appears to be due to congestion, hemorrhage and edema in the lungs. Consideration of dosages required and times until onset of action suggests that, dependent upon the route of administration, the effect is either mediated via a central action or is due to a direct effect on the cardiac and/or respiratory system in the periphery. The pattern and extent of phospholipid hydrolysis in various brain regions was similar following intraventricular injection of the two phospholipases so that no relationship between phospholipid hydrolysis and lethal potency could be established. Concentrations of 5 and 10 μmg/ml of the N. nigricollis and H. haemachatus phospholipases, respectively, were required to block electrical activity of the isolated single electroplax. The ultrastructural changes produced by both phospholipases were also similar. Parallel to the somewhat greater potency on the electroplax, N. nigricollis phospholipase produced slightly greater overall hydrolysis in the innervated and non-innervated membranes of the electroplax than did H. haemachatus phospholipase. The results suggest that these two phospholipases do not have a specific junctional effect and that the small difference in potency on the junction cannot be responsible for the large difference in lethality observed in mammalian species.     

Effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom,on the viability of cells infected with dengue virus

Dengue fever is considered a major public health problem in tropical and subtropical regions. Our study analyzed the effect of BaltPLA_2, a phospholipase A₂ from Bothrops alternatus snake venom, on the viability of cells infected with Dengue virus. In presence of BaltPLA₂, the viability of infected cells increased significantly in virucidal, post-treatment, and adsorption assays. Although preliminary these results reveal the need for further studies to investigated whether BaltPLA₂ has antiviral activity against Dengue virus.     

Phospholipid hydrolysis in serum lipoproteins by a basic phospholipase A2 from Naja nigricollis snake venom and an acidic phospholipase A2 from Naja naja atra snake venom

Apparent Km and Vmax values for PC and PE hydrolysis were determined following exposure of HDL, LDL, and VLDL to a basic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. n. atra snake venom. Both enzymes hydrolyzed the lipoprotein phospholipids approximately as fast as they hydrolyzed pure phospholipids in mixed micelles, however, the N. nigricollis enzyme, which has a much stronger anticoagulant effect than the N. n. atra enzyme, had lower apparent Vmax values. These values were highest for phospholipids in VLDL and lowest for HDL, however, the differences between the lipoproteins were relatively small with the N. nigricollis enzyme while the differences were much larger with the N. n. atra enzyme. Fractions of the two enzymes in which varying numbers of lysines were carbamylated showed much larger differences in relative rates of phospholipid hydrolysis in HDL, LDL and VLDL. Triton X-100 eliminates these differences in rates of hydrolysis. These results are discussed in terms of the differences in the organized structure of the lipoprotein classes and in the penetration ability of the phospholipases.     

A study of cardiotoxic principles from the venom of Bungarus fasciatus (Schneider).

Two cardiotoxin-like fractions (VI A and VI B) were isolated from the venom of Bungarus fasciatus (Schneider) by means of column chromatography on CM cellulose. The homogeneity of these two fractions was verified by microzone and disc electrophoresis. These two fractions shared with cobra cardiotoxin (CTX) many pharmacological actions. Both toxins produced skeletal muscle contracture, depression on cardiac muscle, blockade of neuromuscular transmission, local irritation and had anticholinesterase activity. However, the molecular weights of VI A and VI B were about two times that of CTX and the amino acid composition was also quite different from that of CTX. Moreover, both VI A and VI B were devoid of direct hemolytic action.The contracture inducing activity of VI B and CTX could be abolished by EDTA or high Ca²⁺ (12 mM) and accelerated by low Ca²⁺ (0.5 mM) medium. However, a high Mg²⁺ (10 mM) medium inhibited VI B induced contracture but accelerated CTX contracture. Spermine, spermidine and protamine were without effect on VI B contracture but accelerated profoundly CTX induced contracture. From these results, it is considered that the mode of contracture inducing activity of VI B may be different from that of CTX.     

Relaxant effect of phospholipase A2 from Vipera russelli snake venom on rat aorta

The effect of the acidic phospholipase A2 (PLA2) from Vipera russelli venom on the rat aortic ring was studied and compared with that of acetylcholine (ACh). PLA2 induced relaxation of the aortic ring precontracted with noradrenaline (NA) in a dose-dependent manner. Removal of the endothelium did not reduce the relaxant effect of PLA2. Replacement of Ca2+ by Sr2+ in the medium to inhibit the PLA2 enzyme activity reduced the relaxant effect. Atropine, a muscarinic receptor antagonist, did not affect the relaxant response. The cyclooxygenase inhibitor indomethacin, when equilibrated for 50 min, potentiated the relaxation. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) partially reduced the relaxation. This relaxation was also partially reduced by the guanylate cyclase inhibitor methylene blue. In contrast, the relaxation elicited by ACh was abolished by de-endothelialization, atropine, NDGA or methylene blue. 6-keto-PGF1 alpha (degradation product of prostacyclin) and PGE2 produced by aortic rings were measured by radioimmunoassay. PLA2 (3 X 10(-6) g/ml) increased the output of 6-keto-PGF1 alpha about 10-fold. The production of PGE2 was also increased but to a lesser extent. ACh also increased the output of 6-keto-PGF1 alpha and PGE2. However, prostacyclin released by PLA2 and ACh appears not to contribute to the relaxant effect, since prostacyclin does not relax the rat aorta. It is concluded that the relaxation elicited by PLA2 in the rat aorta is endothelium-independent and partially mediated by lipoxygenase product(s) and cyclic GMP whereas the relaxation induced by ACh was endothelium-dependent, mediated by lipoxygenase product(s) and cyclic GMP, and blocked by atropine.     

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—I: Enzymatic activity on free and membrane bound substrates

The purified phospholipase A₂ of Naja nigricollis venom is a basic, relatively toxic protein, while the purified phospholipase A₂ of Hemachatus haemachatus is neutral and relatively non-toxic. In order to establish whether the difference in toxicity correlates with hydrolytic ability, we compared the two enzymes using substrates in various physical states such as mixed micelles, native soluble lipoprotein or organized in membranes. The purified phospholipids were used as mixed micelles with Triton X-100. When compared on purified egg l-α-phosphatidylcholine (PC), the two enzymes showed similar pH-and temperature-dependence and were equally affected by activators and inhibitors. N. nigricollis phospholipase A₂ had a V_max of 250 μ-equiv. per min per mg and a K_m of 4.2 mM, while H. haemachatus phospholipase A₂ had a V_max of 1052 μ-equiv. per min per mg and a K_m of 2.2 mM. Both enzymes favored the substrates in the liquid-crystalline state. With a buffered egg yolk dilution as substrate, a V_max of 356 μ-equiv. per min per mg and a K_m of 29 mM were found for N. nigricollis, while H. haemachatus had a V_max of 616 μ-equiv. per min per mg and a K_m of 25 mM. The hydrolysis of purified PC, l-α-phosphatidylethanolamine (PE), l-α-phosphatidylserine (PS) and l-α-phosphatidylinositol (PI) was followed with the substrates taken either singly or in various combinations. Significant differences in preference of the two enzymes were apparent on single substrates, such as the comparatively high hydrolysis of PC by H. haemachatus phospholipase A₂ and of PE by N. nigricollis phospholipase A₂. On mixtures of the four substrates, taken either in equal amounts or in proportions resembling the phospholipid distribution of human red cells, rat brain or electric eel Sachs organ, the sequence of substrate preference exhibited by the two enzymes was again strikingly different. A main feature of the N. nigricollis phospholipase A₂ was its high ability to hydrolyze PS. There was no essential difference between the actions of the two enzymes on fresh human red cells. However, erythrocytes from stored, outdated blood were hemolyzed and phospholipids were fully hydrolyzed by N. nigricollis phospholipase A₂, while the H. haemachatus enzyme was nonhemolytic and induced only limited hydrolysis. The same disparity in behavior could be demonstrated on fresh guinea pig erythrocytes. A comparison of hydrolysis, in permeable red cell ghosts and in Triton-solubilized membranes, by the phospholipases, revealed that the high preference of N. nigricollis enzyme for PS was masked by sequestration of this phospholipid within the ghosts.     

Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom

A new phospholipase A2 (PLA2) isoenzyme was isolated from Lachesis muta crude venom, and was named LM-PLA2-II. This enzyme was purified by gel filtration on a Sephacryl S-200 HR column followed by reverse-phase chromatography on a C2/C18 column. LM-PLA2-II consists of a single polypeptide chain with an apparent molecular mass of 18 kDa and an isoelectric point at pH 5.4. The amino terminal sequence of the enzyme revealed a high degree of homology with other PLA2s from several sources. LM-PLA2-II has a high indirect hemolytic activity and a potent inhibitory effect on platelet aggregation induced by ADP and collagen. It also produces a significant paw edema reaction in rats. The edematous response in rats was abolished by pretreatment with either indomethacin or dexamethasone, suggesting the involvement of cyclo-oxygenase. Pretreatment of LM-PLA2-II with p-bromophenacyl bromide abolished all of these actions, clearly indicating that the biological activities, including the edematogenic effect, are dependent entirely on its enzymatic activity.     

Effects of morin on snake venom phospholipase A2 (PLA2).

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.     

Revised amino acid sequences of the three major phospholipases A2 from Bungarus fasciatus (banded krait) venom

C.-S. , J.-M. , C.-H. , S.-W. , I.-H. , H.-S. and T.-B. . Revised amino acid sequences of the three major phospholipases A₂ from Bungarus fasciatus (banded krait) venom. Toxicon 28, 1457–1468, 1990.—The structures of three cardiotoxin-like proteins obtained from the venom of Bungarus fasciatus (banded krait) were elucidated previously ( and , 1980, 1981). Since their molecular sizes are similar to that of phospholipase A₂ and since they show weak phospholipase A₂ activities ( et al., 1983), a further study of their primary structures was carried out. Fractions Va, Vb-2 and VI, corresponding to the former V-2, V-3 and VI were determined to be typical phospholipases A₂. Among 118 amino acid residues, they all have in common a Pro 29 between Gly 28 and Gly 30, the latter two residues being implicated in Ca²⁺ binding together with Tyr 26 and Asp 47.     

Purification and characterization of a phospholipase A2 from Cerastes cerastes (horn viper) snake venom

A single phospholipase A2 has been found in Cerastes cerastes venom, purified to homogeneity by a combination of chromatographic steps involving gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sephadex A-50. Its mol. wt, its amino acid composition and its partial amino acid sequence have been determined. High homologies between its sequence and those of other Viperid phospholipides A2 have been noticed. The phospholipase was non-lethal to mice up to a dose as high as 25 mg/kg by i.p. and i.v. injection. This non-toxic enzyme exhibited an acidic isoelectric point and hydrolyzed monolayers of different short chain phospholipids. Some kinetic parameters have been studied potentiometric titration (with or without Triton X-100) and the rate of catalysis seemed not to be affected by changes in the physical state of the substrate.     

Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A(2) from Bothrops asper snake venom.

Myotoxic class II phospholipases A(2) from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115-129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1-15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A(2), showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins (Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A(2), regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.     

Neurotoxic activity of Gln49 phospholipase A(2) from Gloydius ussuriensis snake venom

A novel neurotoxic protein phospholipase A(2) (PLA(2)), molecular weight 13 881.83 Da, has been isolated from snake venom of Gloydius ussuriensis, named as Gln49-PLA(2), which shows weak lethal toxic, myotoxic and apparent anticoagulant activity, but lacks phospholipase activity. The Gln49-PLA(2) obviously induced an increase of the pain threshold in intoxicated 615 mice compared with the control group, suggesting it is a neurotoxin. Hot-plate tests also showed that its analgesic activity was dose-dependent, and naloxone antagonized the analgesic effect, implying the mechanism of action of Gln49-sPLA(2) is correlated with opioid receptors. Electrophysiology studies revealed decreases in the action potential and the nerve conduction velocity in isolated hoptoad (Bufo bufo gargarizans Cantor) sciatic nerve, indicating Gln49-PLA(2) most probably had effects on ion channels.     

A novel phospholipase A(2) from the venom glands of Bungarus candidus: cloning and sequence-comparison.

The presence of phospholipase A(2) (PLA(2)) in the venom of Malayan krait (Bungarus candidus) and its structure were studied. The PLA(2) cDNAs from the venom gland of B. candidus (Indonesia origin) were amplified by the polymerase chain reactions (PCR) and cloned. The primers used were based on the cDNA sequences of several homologous B. multicinctus venom PLA(2)s. In addition to the A-chains of beta-bungarotoxins, a novel B. candidus PLA(2) was cloned and its full amino acid sequence deduced. Having totally 125 amino acid residues, the PLA(2) contains a pancreatic loop and is 61% identical to the acidic PLA(2) of king cobra venom. However, the enzyme was not detected from the venom sample. Its structural relationships to other elapid venom PLA(2)s were analyzed with a phylogenetic tree and discussed.     

Phospholipase A2 activity of long-chain cardiotoxins in the venom of the banded krait (Bungarus fasciatus)

Reinvestigation of the long-chain cardiotoxins from Bungarus fasciatus venom reveals that they are weak phospholipases of the A₂ type. The specific activities (units/mg) toward egg lecithin are 0.42, 1.65, and 0.29 for the long-chain cardiotoxins V-2, V-3 and VI, respectively.