Phospholipases: melittin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes.

Melittin isolated from the venom of the common honey bee is a potent activator for bee venom phospholipase A₂-catalyzed hydrolysis of unsonicated liposomes of egg phosphatidyl choline. At 37 °C and pH 8, the rate of this enzymatic reaction is increased approximately 300-fold by the addition of 8 × 10^?5m melittin. The magnitude of facilitation of the phospholipase A₂ reaction is much greater than that previously reported by other workers for systems involving sonicated egg phosphatidyl choline liposomes or Escherichia coli membrane fragments as substrates. Melittin having lysines quantitatively modified through reaction with methyl acetimidate is as effective a potentiator of phospholipase A₂ activity as the unmodified material. The same result was obtained for melittin in which the single tryptophan residue was modified. Melittin modified by succinylation retained approximately 50% of its capacity to facilitate phospholipase A₂ activity. In contrast, a modified melittin in which the C-terminal four amino residues were removed, acetimidated des(23–26)melittin, is a very poor activator, as is a mixture of this peptide with the C-terminal tetrapeptide. In contrast to the results with egg lecithin liposomes, melittin has little influence on the susceptibility of monomolecular aqueous solutions of dihexanoylphosphatidyl choline to phospholipase A₂ attack.     

Action of phospholipases on the cytoplasmic membrane of Escherichia coli. Stimulation by melittin

The emission maximum of the single tryptophan residue of melittin was measured in the presence of phosphatidylethanolamine liposomes and Escherichia coli cytoplasmic membranes. In both cases, the fluorescence maximum was shifted to shorter wavelengths indicating a transfer of the indole ring to an apolar environment. E. coli membranes were labelled in position 2 of their phospholipids with [¹⁴C]oleic acid. These membranes were used for measuring the activity of an endogenous phospholipase A₂. A slow hydrolysis is observed, which can be accelerated by adding melittin. The extent of the stimulation depends on the molar ratio of melittin to membrane phospholipid. Under suitable conditions, the initial rate of hydrolysis is six to seven times higher in the presence than in the absence of melittin. The action of the phospholipase A₂ from bee venom is also stimulated by melittin. An identical stimulation was observed with either E. coli membranes or pure phosphatidylethanolamine liposomes as substrate.     

The influence of melittin on the rotation of band 3 protein in the human erythrocyte membrane

The rotational mobility of band 3, a protein constituent of the human erythrocyte membrane, was measured by observing the flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, a pharmacologically active polypeptide from honey bee (Apis mellifera) venom, a dose-dependent loss of rotational mobility was detected. With acetylated melittin, the ability to immobilise is reduced. Succinylated melittin, however, is devoid of immobilising activity.The possible relevance of these findings to the normal mode of action of melittin was examined by comparing the relative abilities of the native, acetylated and succinylated melittins to lyse erythrocytes and synergise with phospholipase A₂, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin > acetyl melittin > succinyl melittin = 0, the same as their order of effectiveness in immobilising band 3.A mechanism is proposed in which melittin is anchored in the membrane by its hydrophobic N-terminus, while its cationic C-terminal moiety binds to negatively charged residues on membrane proteins. This leads either directly or indirectly to protein aggregation and hence loss of mobility. From a detailed comparison of the different effects of the melittin derivatives, it is concluded that melittin may function in vivo by aggregating membrane proteins in order to allow phospholipase A₂ to gain access to the membrane bilayer and commence catalysis.     

Selective solubilization by melittin of glycophorin A and acetylcholinesterase from human erythrocyte ghosts

Melittin, the main basic and hydrophobic peptide of bee venom, has been used for solubilizing membrane components of the human erythrocyte ghost. Up to 1.0 mM, it does not extract any phospholipid. Between 0.1 and 1.0 mM, it solubilizes partially glycophorin A and acetylcholinesterase. When the membrane is first degraded by phospholipase A₂, the solubilization of both proteins by melittin is total, and 48% of the phospholipids are removed, mainly as lysoproducts, whereas phospholipase A₂, by itself, has no solubilizing properties. In its melittin-solubilized state, acetylcholinesterase is in a dimeric form and displays a slow time-dependent irreversible inactivation. Triton X-100 at 1.0% (v/v) interrupts the inactivation. We suggest that melittin binds to the hydrophobic site of acetylcholinesterase which anchors it in the lipid bilayer.     

Allergens of honey bee venom.

The allergenic activities of four purified components of honeybee venom were studied by using histamine release from leukocytes of bee sting-allergic patients. The components studied were hyaluronidase, phospholipase A₂, melittin and apamin with molecular weights, respectively, of about 50,000, 15,800, 2840 and 2038 d. In six of the seven patients studied, hyaluronidase and phospholipase were, respectively, on the average about two and eight times more active by weight than the venom. The situation was reversed in one patient in that hyaluronidase and phospholipase A₂ were, respectively, 90 and 0.5 times more active than the venom. With this single exception, hyaluronidase and phospholipase were about equally active on a molar basis as allergens. Melittin was on the average about one-tenth as active as the venom, and apamin was inactive as an allergen.Chemical modifications of phospholipase A₂ were carried out. Succinylation of eight of its eleven amino groups yielded a derivative that retained 4% of the enzymic activity of the native enzyme. Reduction and carboxymethylation of its four disulfide bonds or cyanogen bromide cleavage of its three methionyl bonds yielded enzymatically inactive derivatives. These derivatives showed varying decreases of allergenic activities when compared to the native enzyme. The results indicate that the antigenic determinants of phospholipase depend on the charge, the amino acid sequence and the conformation of the molecule.     

Single-molecule phospholipase A2 becomes processive on melittin-induced membrane deformations

While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A₂ (PLA₂), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA₂ activity is 25-fold higher. Tracking of single-molecule PLA₂ revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA₂ work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.     

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.     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.     

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A₂, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A₂ can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.     

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.     

Isolation and characterization of the major phospholipase a2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

• 1.1. The major phospholipase A₂ (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
• 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
• 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
• 4.4. The phospholipase A₂ exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

     

Calcium-dependent binding of calmodulin to phospholipase A2 subunits induces enzymatic activation

Calmodulin interacted with phospholipase A₂ from two different sources, as established by affinity chromatography, dimethylsuberimidate protein crosslinking, and phospholipase A₂ assays. Calmodulin was covalently crosslinked to pancreatic and bee venom phospholipases A₂ in a calcium-dependent manner, and enhanced the enzymatic activities of these phospholipases. Pancreatic phospholipase A₂ was separated into two species of identical molecular weight by calmodulin affinity chromatography; the species that bound to immobilized calmodulin in a calcium-dependent manner was stimulated by calmodulin. This presents further evidence that phospholipase A₂ is directly activated by calmodulin.     

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.     

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.     

Hemorrhagic principles in the venom of Bitis arietans,a viperous snake. I. Purification and characterization

Two hemorrhagic principles (Bitis arietans hemorrhagin a and b: abbreviated as BHRa and BHRb) were purified from the venom of the viperous snake Bitis arietans (puff adder) by gel filtration, ion-exchange and absorption chromatography. A 10-fold purification was achieved for BHRa and 7-fold for BHRb with an overall yield of 6.4% of hemorrhagic activity. The hemorrhagins were homogeneous according to disc- and SDS-polyacrylamide gel electrophoresis and immunodiffusion. BHRa and BHRb consist of 623 and 685 amino-acid residues and their apparent molecular weights were 68 000 and 75 000, respectively. They were also immunologically distinct. The purified hemorrhagins express proteolytic activity with heat-denatured casein and hide powder azure. The proteolytic activity with heat-denatured casein was almost the same as that of the crude venom, but that with hide powder azure was less than one-tenth of that of the crude venom. The purified hemorrhagins were free of arginine esterase and phospholipase A₂ activities and they are acid labile hemorrhagic toxins. Their hemorrhagic activity was inhibited by EDTA, cysteine and by polyvalent anti-snake serum, but not by phenylmethanesulfonyl fluoride or soybean trypsin inhibitor.     

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization,isolation of a myotoxic phospholipase A2 homologue and neutralization by two antivenoms

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A₂ activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A₂ homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A₂ variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.     

Effects of petrosaspongiolide R on the surface topology of bee venom PLA(2): a limited proteolysis and mass spectrometry analysis

Petrosaspongiolides are sponge metabolites belonging to the family of the γ-hydroxybutenolide marine terpenoids. They possess a remarkable in vitro and in vivo anti-inflammatory profile, due to the specific inhibition of group II and III secretory phospholipase A₂ enzymes, and for this reason can be considered as potential lead for the development of anti-inflammatory drugs. The molecular mechanism of bee venom phospholipase A₂ inactivation has been identified, and the ligand-enzyme complex formation is guided by either non-covalent and covalent interactions. In this work we have analyzed the conformational changes induced by petrosaspongiolide R on the bee venom phospholipase A₂ topology during the molecular recognition process, through the application of limited proteolysis and mass spectrometric methodologies. The results are indicative of structural changes at the N- and C-terminal domains producing a more compact conformational arrangement of the enzyme.     

The synergism of cardiotoxin and phospholipase A2 in hemolysis

The synergistic effect of exogenous cobra phospholipase A₂ on the hemolysis rate of guinea pig erythrocytes by highly purified snake venom cardiotoxins was investigated. In the presence of phospholipase A₂ the reaction was not only faster and had a lower activation energy but followed a sigmoidal instead of a linear time course. Similar results were obtained using porcine pancreatic phospholipase A₂. Significantly, addition of even a trace of cobra phospholipase A₂ (approx. 0.1%, w/w) was sufficient to bring about the full synergistic effect, emphasizing the stringent purity requirements for any meaningful investigation of cardiotoxin's own action. The possibility that the action of cardiotoxin on its own may involve the stimulation of an endogenous phospholipase is discussed in the light of the results obtained with exogenous cobra enzyme.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.