melitten and mastoparan

While knowledge of the composition and mode of action of bee and wasp venoms dates back 50 years, the therapeutic value of these toxins remains relatively unexploded. The properties of these venoms are now being studied with the aim to design and develop new therapeutic drugs. Far from evaluating the extensive number of monographs, journals and books related to bee and wasp venoms and the therapeutic effect of these toxins in numerous diseases, the following review focuses on the three most characterized peptides, namely melittin, apamin, and mastoparan. Here, we update information related to these compounds from the perspective of applied science and discuss their potential therapeutic and biotechnological applications in biomedicine.

Topics: Animals; Apamin; Humans; Intercellular Signaling Peptides and Proteins; Melitten; Peptides; Wasp Venoms

The physico-chemical and biological properties of cytolytic peptides derived from diverse living entities have been discussed. The principal sources of these agents are bacteria, higher fungi, cnidarians (coelenterates) and the venoms of snakes, insects and other arthropods. Attention has been directed to instances in which cytolytic peptides obtained from phylogenetically remote as well as from related sources show similarities in nature and/or mode of action (congeneric lysins). The manner in which cytolytic peptides interact with plasma membranes of eukaryotic cells, particularly the membranes of erythrocytes, has been discussed with emphasis on melittin, thiolactivated lysins and staphylococcal alpha-toxin. These and other lytic peptides are characterized in Table III. They can be broadly categorized into: (a) those which alter permeability to allow passage of ions, this process eventuating in colloid osmotic lysis, signs of which are a pre-lytic induction or latent period, pre-lytic leakage of potassium ions, cell swelling and inhibition of lysis by sucrose. Examples of lysins in which this mechanism is involved are staphylococcal alpha-toxin, streptolysin S and aerolysin; (b) phospholipases causing enzymic degradation of bilayer phospholipids as exemplified by phospholipases C of Cl. perfringens and certain other bacteria; (c) channel-forming agents such as helianthin, gramicidin and (probably) staphylococcal delta-toxin in which toxin molecules are thought to embed themselves in the membrane to form oligomeric transmembrane channels.

Topics: Alamethicin; Animals; Ant Venoms; Arthropod Venoms; Bacterial Proteins; Bacterial Toxins; Basidiomycota; Cell Membrane; Cnidarian Venoms; Coleoptera; Cytotoxins; Erythrocyte Membrane; Gramicidin; Hemolysin Proteins; Intercellular Signaling Peptides and Proteins; Macromolecular Substances; Marine Toxins; Melitten; Microscopy, Electron; Mycotoxins; Peptides; Phospholipase D; Phospholipases A; Pore Forming Cytotoxic Proteins; Protein Conformation; Scyphozoa; Snake Venoms; Streptolysins; Sulfhydryl Compounds; Type C Phospholipases; Vibrio; Wasp Venoms

Cathelicidins are phylogenetically ancient, pleiotropic host defense peptides-also called antimicrobial peptides (AMPs)-expressed in numerous life forms for innate immunity. Since even the jawless hagfish expresses cathelicidins, these genetically encoded host defense peptides are at least 400 million years old. More recently, cathelicidins with varying antipathogenic activities and cytotoxicities were discovered in the venoms of poisonous snakes; for these creatures, cathelicidins may also serve as weapons against prey and predators, as well as for innate immunity. We report herein the expression of orthologous cathelicidin genes in the venoms of four different South American pit vipers (Bothrops atrox, Bothrops lutzi, Crotalus durissus terrificus, and Lachesis muta rhombeata)-distant relatives of Asian cobras and kraits, previously shown to express cathelicidins-and an elapid, Pseudonaja textilis. We identified six novel, genetically encoded peptides: four from pit vipers, collectively named vipericidins, and two from the elapid. These new venom-derived cathelicidins exhibited potent killing activity against a number of bacterial strains (S. pyogenes, A. baumannii, E. faecalis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa), mostly with relatively less potent hemolysis, indicating their possible usefulness as lead structures for the development of new anti-infective agents. It is worth noting that these South American snake venom peptides are comparable in cytotoxicity (e.g., hemolysis) to human cathelicidin LL-37, and much lower than other membrane-active peptides such as mastoparan 7 and melittin from bee venom. Overall, the excellent bactericidal profile of vipericidins suggests they are a promising template for the development of broad-spectrum peptide antibiotics.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Bothrops; Cathelicidins; Hemolysis; Humans; Immunity, Innate; Intercellular Signaling Peptides and Proteins; Melitten; Peptides; Species Specificity; Venoms; Wasp Venoms

At present, colistin is among the few antibiotics effective against Acinetobacter baumannii clinical isolates. However, in the last few years, colistin-resistant A. baumannii strains have been isolated. Therefore, antibiotics effective against these usually pan-resistant colistin-resistant A. baumannii strains are required. The main objective of this study was to analyse the activity of 15 peptides against colistin-susceptible and colistin-resistant A. baumannii. The MICs were determined by microdilution. Among these 15 antimicrobial peptides (AMPs), melittin, indolicidin and mastoparan showed good activity against both colistin-susceptible and colistin-resistant A. baumannii. Further studies of mastoparan with time-killing curves showed bactericidal activity at MIC ×8 for both colistin-susceptible and colistin-resistant A. baumannii. In conclusion, mastoparan may be a potential alternative for the treatment of colistin-resistant A. baumannii infections.

Topics: Acinetobacter baumannii; Acinetobacter Infections; Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Colistin; Drug Resistance, Bacterial; Inhibitory Concentration 50; Intercellular Signaling Peptides and Proteins; Melitten; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Time Factors; Wasp Venoms

Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein-protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein-protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin-melittin and calmodulin-mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin-melittin and three interpeptide cross-links for calmodulin-mastoparan. Using cross-linking sites of calmodulin-mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. We also demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.

Topics: Amino Acid Sequence; Calmodulin; Cross-Linking Reagents; Intercellular Signaling Peptides and Proteins; Mass Spectrometry; Melitten; Molecular Sequence Data; Peptides; Protein Binding; Protein Conformation; Protein Interaction Mapping; Succinimides; Wasp Venoms

The dependence of membrane properties on their composition was studied by following the adhesion and spreading of unilamellar and multilamellar liposomes on static mercury electrodes with the help of chronoamperometry. The analysis of the peak-shaped signals allows determining the kinetic parameters of the three-step adhesion-spreading process. The presence of cholesterol in the membrane stabilizes the bilayer in the liquid-crystalline phase, and destabilizes the gel phase. The kinetic parameters also show the effect of superlattice formation in the DMPC-cholesterol system. The detergent triton X-100 is only incorporated in the liquid-crystalline DMPC membranes, and it is expelled to the solution when the membrane is transformed to the gel phase. In the liquid-crystalline membrane, it enhances the adhesion-spreading of liposomes on mercury. The lytic peptides mastoparan X and melittin affect the adhesion-spreading in a similar manner. For the rupture-spreading step, their effect is explained by pore formation. The results obtained with lecithins of different length suggest that the bilayer opening process has much in common with flip-flop translocations. For this process the activation energies were found to be independent of the chain length of the lecithin molecules, while the preexponential factor in the Arrhenius equation decreases drastically for longer chains.

Topics: Adhesiveness; Cholesterol; Dimyristoylphosphatidylcholine; Electrochemistry; Electrodes; Intercellular Signaling Peptides and Proteins; Kinetics; Lecithins; Liposomes; Melitten; Mercury; Octoxynol; Peptides; Phase Transition; Porosity; Thermodynamics; Wasp Venoms

A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the L-citrulline formation of nNOS from L-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.

Topics: Allosteric Regulation; Animals; Binding Sites; Calmodulin; Citrulline; Cytochrome Reductases; DNA, Complementary; Enzyme Inhibitors; Intercellular Signaling Peptides and Proteins; Kinetics; Melitten; Nitric Oxide Synthase Type I; Peptides; Rats; Recombinant Proteins; Wasp Venoms

Our recent data implicated small molecular weight G-proteins (e.g., H-Ras) in interleukin 1beta (IL 1beta)-induced metabolic dysfunction and apoptotic demise of the islet beta cell (Tannous et al., Biochem Pharmacol 2001; 62:1459-1468, Kowluru and Morgan, Biochem Pharmacol, 2002; 63:1027-1035, Chen et al. Biochem Pharmacol, 2003; 66:1681-1694). Recently, we have shown that mastoparan, a tetradecapeptide from wasp venom, has been shown to directly activate islet endogenous G-proteins and regulate islet function (Amin et al., Endocrinology 2003; 144: 4508-4518). Herein, we investigated potential contributory roles, if any, of mastoparan (Mas)-sensitive G-proteins in IL-induced nitric oxide (NO) release from insulin-secreting HIT-T15 cells. While, ineffective by itself, Mas significantly potentiated IL-induced NO release from HIT-T15 cells. Interestingly, Mas-17, an inactive analog of Mas, also potentiated IL-induced NO release, suggesting that the potentiating effect of Mas may not involve activation of specific G-proteins. Such potentiating effects on IL-induced NO release were also demonstrable in the presence of another polycationic compound, melittin. Together, these findings suggest that Mas-induced potentiation of IL-induced NO release may in part be due to its amphiphilic and polycationic nature. These data also warrant caution in the use of Mas to study its regulation of cellular function without the use of an appropriate negative control, such as Mas-17.

Topics: Dose-Response Relationship, Drug; GTP-Binding Proteins; Humans; Insulin; Intercellular Signaling Peptides and Proteins; Interleukin-1; Islets of Langerhans; Melitten; Nitric Oxide; Nitrites; Peptides; Wasp Venoms

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

Topics: Binding, Competitive; Cholera Toxin; Fabaceae; Guanosine 5'-O-(3-Thiotriphosphate); Heterotrimeric GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Lipopolysaccharides; Melitten; Monomeric GTP-Binding Proteins; Oligosaccharides; Peptides; Pertussis Toxin; Plant Roots; Rhizobium; Signal Transduction; Sulfur Radioisotopes; Symbiosis; Wasp Venoms

Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of alpha-helical, cationic peptides. For the first time, a comparison of the primary structures of alpha-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x)3KxWxS(x)5L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a pore-forming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 micro m), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.

Topics: Africa, Southern; Amino Acid Sequence; Animals; Antifungal Agents; Antimicrobial Cationic Peptides; Fungi; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Melitten; Molecular Sequence Data; Molecular Weight; Peptides; Protein Structure, Secondary; Scorpion Venoms; Sequence Homology, Amino Acid; Wasp Venoms

A GTP-binding protein (G-protein), termed G-exocytosis (Ge), mediates the effects of calcium ions in the late stages of the adrenocorticotrophin (ACTH) secretory pathway. An activator of Ge, mastoparan, also stimulates phospholipase A(2) and so a comparison of other phospholipase A(2)-activating peptides, melittin and phospholipase A(2)-activating peptide was made with mastoparan to assess whether phospholipase A(2)activation was an important component of Ge-evoked secretion. All three peptides stimulated ACTH secretion in the effective absence of calcium ions from permeabilised cells, actions potentiated by a phospholipase A(2)inhibitor. Ca(2+)-evoked secretion from permeabilised cells was similarly potentiated by a phospholipase A(2) inhibitor. Furthermore, arachidonic acid inhibited Ca(2+)- and Ge-evoked ACTH secretion, an action blocked by the cyclo-oxygenase inhibitor ibuprofen. This study suggests that the products of phospholipase A(2)-generated arachidonic metabolism may exert an inhibitory action on the late post-Ca(2+) stages of the ACTH secretory pathway and that prostaglandins may be the active agents in this capacity.

Topics: Adrenocorticotropic Hormone; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Cell Membrane Permeability; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Ibuprofen; Intercellular Signaling Peptides and Proteins; Melitten; Peptides; Phospholipases A; Proteins; Tumor Cells, Cultured; Wasp Venoms

Mono-Mac-6 (MM6) human monocytes ingest IgG-opsonized particles better than other human cell lines. We compared the phagocytic signaling pathway in MM6 with human monocytes. MM6 expressed FcgammaRI at levels similar to monocytes, whereas FcRgammaII expression was approximately double. MM6 ingested IgG-opsonized erythrocytes (EIgG) in a calcium-independent manner. Incubation of MM6 with bromoenol lactone, an inhibitor of the phagocytic phospholipase (pPL), coordinately decreased phagocytosis and pPL activity. This inhibition was overcome by exogenous arachidonic acid, suggesting that phagocytosis requires pPL activation and arachidonic acid release. MM6 phagocytosis was inhibited with staurosporine and activated with diacylglycerol, supporting a role for protein kinase C (PKC) in this process. The pPL activators mastoparan and melittin restored phagocytosis to PKC-inhibited cells, suggesting that pPL lies downstream from PKC. These results suggest that the MM6 signal transduction pathway for IgG-mediated phagocytosis is similar to that of monocytes (PKC-->pPL-->arachidonic acid-->phagocytosis). The results are discussed in the context of the finding that MM6 exhibit low phagocytosis relative to monocytes and thus may represent an attractive cell line for molecular manipulation in "recovery of function" studies.

Topics: Arachidonic Acids; Cell Line; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Group VI Phospholipases A2; Humans; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Macrophages; Melitten; Monocytes; Opsonin Proteins; Peptides; Phagocytosis; Phospholipases A; Protein Kinase C; Receptors, IgG; Rosette Formation; Wasp Venoms

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction.

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Animals; Calcium-Binding Proteins; Calmodulin; Calorimetry; Circular Dichroism; Entamoeba histolytica; Escherichia coli; Hydrogen-Ion Concentration; Intercellular Signaling Peptides and Proteins; Melitten; Models, Molecular; Molecular Sequence Data; Peptides; Protein Binding; Protein Structure, Secondary; Recombinant Proteins; Sequence Homology, Amino Acid; Thermodynamics; Wasp Venoms

Some basic amphiphilic peptides are known to directly stimulate heterotrimeric GTP-binding proteins (G proteins). Mastoparan and melittin are known to stimulate Gi activities. Here, we found melittin inhibited guanine nucleotide-dependent adenylyl cyclase activity in synaptic membranes of the rat cerebral cortex. However, in insect cell membranes overexpressing specific heterotrimeric G proteins using baculovirus expression system, melittin showed unique effects different from those by mastoparan on G protein activities. This peptide markedly stimulated Gi1 and G11 activities, whereas it did inhibit Gs activities. Kinetic studies revealed that the inhibition of Gs activity by melittin is attributed to the inhibition of GDP release in exchange for added guanine nucleotides (or the association of guanine nucleotides). Thus, melittin may be the first metabostatic peptide inhibiting G protein (Gs) activity, and both mechanisms through the stimulation of Gi and inhibition of Gs might be involved in the melittin-induced inhibition of adenylyl cyclase.

Topics: Adenylyl Cyclases; Animals; Brain; Cell Membrane; Cells, Cultured; Enzyme Activation; GTP-Binding Protein alpha Subunits, Gs; Intercellular Signaling Peptides and Proteins; Male; Melitten; Membrane Proteins; Peptides; Rats; Rats, Wistar; Recombinant Proteins; Spodoptera; Synaptic Membranes; Wasp Venoms

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.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Arachidonic Acid; Calcitriol; Cell Differentiation; Chondrocytes; Enzyme Activation; Growth Plate; Intercellular Signaling Peptides and Proteins; Male; Melitten; Peptides; Phospholipases A; Phospholipases A2; Protein Kinase C; Rats; Rats, Sprague-Dawley; Wasp Venoms

The effect of the alpha-helical polycationic peptides magainin 2, melittin, mastoparan and cecropin on the viability of Brucella abortus 544 (type species), B. abortus S19 (vaccine strain) and B. abortus S2308 (vaccine challenge strain) was determined. Rough mutants of these strains and the rough candidate vaccine strain B. abortus RB51 were also tested. S. typhimurium was used as a control. The peptides did not affect the viability of B. abortus smooth strains but some of the peptides affected viability of the rough strains. Magainin 2 at a concentration of 100 micrograms ml-1 did not reduce the viability of the rough B. abortus strains. Cecropin at a concentration of 15 micrograms ml-1 reduced the viability of the rough strains by approximately 10-fold. Mastoparan at a concentration of 50 micrograms ml-1 reduced the viability of the rough strains by approximately 100-fold. Melittin at a concentration of 20 micrograms ml-1 reduced the viability of the rough strains of B. abortus by approximately 1000-fold. The brucellae were significantly more resistant to all the cationic peptides than was S. typhimurium.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Brucella abortus; Intercellular Signaling Peptides and Proteins; Magainins; Melitten; Microbial Sensitivity Tests; Peptides; Wasp Venoms; Xenopus Proteins

We examined the susceptibilities of Bordetella pertussis strains to several antimicrobial peptides by determining the concentration required to inhibit or kill 50% of the bacterial population. The peptides are ranked in decreasing potency as follows: cecropin B > cecropin A >> melittin > cecropin P1 > (ala8,13,18)-magainin II amide > mastoparan = defensin HNP1 > protamine > or = magainin II = magainin I. By using a radial diffusion assay to compare susceptibilities between strains, wild-type B. pertussis BP338 was more resistant than the avirulent bvg mutant strain BP347 and the brk mutant strain BPM2041 to killing by cecropin P1. In contrast, compared with the wild type, the avirulent BP347 strain was highly resistant to killing by protamine and defensin HNP1.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bordetella pertussis; Intercellular Signaling Peptides and Proteins; Melitten; Microbial Sensitivity Tests; Peptides; Wasp Venoms

The following study was undertaken to determine whether an inducible calcium influx pathway is present in intact bloodstream forms of Trypanosoma brucei. Fura-2 fluorescence was used to demonstrate that amphiphilic peptides and amines, including melittin, mastoparan and compound 48/80, each produced a dose dependent calcium influx across the plasma membrane. Calcium influx did not result from general disruption of membrane integrity, since a corresponding influx of ethidium bromide or other divalent cations was not observed. Instead, the calcium influx was selectively blocked by the calcium channel antagonists, La3+, Cd2+ or Ni2+, and was not affected by the Na+ channel antagonists, tetrodotoxin or amiloride. Activation of the trypanosome calcium influx pathway was dependent upon an intact membrane potential, and the rise in intracellular free calcium concentration ([Ca2+]i) was reversed upon membrane depolarization with gramicidin D. Changes in Ins(1,4,5)P3 did not accompany the calcium influx. Overall, these data provide the first evidence of an inducible calcium influx pathway in T. brucei, and describe methods to selectively manipulate this pathway.

Topics: Amines; Animals; Calcium; Cell Membrane Permeability; Inositol 1,4,5-Trisphosphate; Intercellular Signaling Peptides and Proteins; Ion Transport; Melitten; Membrane Potentials; p-Methoxy-N-methylphenethylamine; Peptides; Phosphatidylinositol 4,5-Diphosphate; Trypanosoma brucei brucei; Wasp Venoms

Melittin is known to be a major hydrophobic peptide component in honeybee venom that can cause as much elevation of fertilization membrane of sea urchin eggs as normal fertilization. The action of melittin has been thought to be closely related with its ability to facilitate the phospholipase A2 activity on the eggs. However, another peptide "mastoparan" from wasp venom was not found here to cause any elevation of the membrane, although it can activate the enzyme as well as melittin. On the other hand, mastoparan was found to get the membrane-elevating activity only when its amino groups were modified with hydrophobic substituents. N epsilon-Substituted mastoparan with a dansyl group in Lys11 residue was most effective among the analogs examined here. Our findings indicate that the facilitation of phospholipase by the peptides have little relation with the membrane generation. Such hydrophobic moiety as the dansyl group in the peptides must cause the cortical reaction on the eggs in cooperation with peptide moiety. The dansylated peptide will be a useful tool to induce the artificial fertilization of sea urchin eggs.

Topics: Amino Acid Sequence; Animals; Dose-Response Relationship, Drug; Female; Fertilization; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Melitten; Molecular Sequence Data; Ovum; Peptides; Phospholipases A; Phospholipases A2; Sea Urchins; Structure-Activity Relationship; Wasp Venoms

Previous studies demonstrated that both protein kinase C (PKC) and arachidonic acid (AA) are required for IgG-mediated phagocytosis by human monocytes. We have characterized a calcium-independent "phagocytic" phospholipase A2 (designated pPL) that mediates arachidonic acid release. The present studies were designed to order PKC and pPL in the phagocytic signaling pathway. The PKC inhibitors staurosporine and calphostin C caused a coordinated decrease in phagocytosis of IgG-opsonized erythrocytes and arachidonic acid release. The PLA2 activators mastoparan and melittin restored phagocytosis to PKC-inhibited cells, but were ineffective in monocytes pretreated with the pPL inhibitor bromoenol lactone. Similarly, PKC activation with PMA and diacylglycerol enhanced phagocytosis in the absence, but not in the presence, of bromoenol lactone. These results indicate that pPL may be regulated by an upstream phosphorylation event. Thus, we examined the effects of Ab-opsonized glass bead ingestion, okadaic acid-mediated inhibition of phosphatases, and PMA treatment on the activity of pPL and on its distribution between the cytosolic and membrane-associated compartments. IgG-opsonized erythrocytes and okadaic acid caused an overall increase in pPL activity, with a twofold increase in membrane-associated pPL. PMA treatment caused a 1.8-fold increase in membrane-associated pPL activity. Okadaic acid and PMA mimic IgG-opsonized erythrocytes with respect to membrane activation of pPL, suggesting that pPL activity may be regulated by PKC. Collectively, these results indicate that pPL activity is modulated by PKC during IgG-mediated phagocytosis, and that the PKC requirement can be bypassed by direct activation of pPL.

Topics: Arachidonic Acid; Cells, Cultured; Enzyme Activation; Ethers, Cyclic; Humans; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Melitten; Monocytes; Okadaic Acid; Peptides; Phagocytosis; Phospholipases A; Phospholipases A2; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate; Time Factors; Wasp Venoms

beta-Amyloid protein (A beta) is a member of a small group of proteins that accumulate as amyloid deposits in various tissues. It has recently been demonstrated that the toxicity of A beta toward some neural cells is caused by oxidative damage. Since all of the amyloid diseases are characterized by protein deposited in the antiparallel beta-sheet conformation, it was asked whether there is a common toxic mechanism. It is shown here that the protein components of other human amyloidoses, including amylin, calcitonin, and atrial natriuretic peptide, are all toxic to clonal and primary cells. The toxicity is mediated via a free radical pathway indistinguishable from that of A beta. Experiments with synthetic peptides suggest that it is the amphiphilic nature of the peptides generated by their beta structure rather than their beta structure per se that causes toxicity. These results tend to rule out the alternative that amyloid toxicity is exclusively mediated via specific cell surface receptors.

Topics: Amino Acid Sequence; Amyloid; Amyloid beta-Peptides; Animals; Atrial Natriuretic Factor; Brain Neoplasms; Calcitonin; Cell Line; Cell Survival; Flow Cytometry; Humans; Hydrogen Peroxide; Intercellular Signaling Peptides and Proteins; Islet Amyloid Polypeptide; L-Lactate Dehydrogenase; Melitten; Molecular Sequence Data; NADH, NADPH Oxidoreductases; Oligopeptides; Onium Compounds; p-Methoxy-N-methylphenethylamine; Peptide Fragments; Peptides; Protein Structure, Secondary; Rats; Structure-Activity Relationship; Tumor Cells, Cultured; Wasp Venoms

The ability of the amphiphilic peptides, melittin and mastoparan, to modulate the production of inositol(1,4,5)trisphosphate in cultured plant (Daucus carota L.) cells was investigated. When added to intact cells melittin and mastoparan caused a rapid and dose-dependent increase in inositol(1,4,5)trisphosphate concentrations. In isolated protoplasts, inositol(1,4,5)trisphosphate levels were 12- to 16-fold higher than in the corresponding cells and neither melittin nor mastoparan was able to significantly affect inositol(1,4,5)trisphosphate production. Melittin and mastoparan had a strong inhibitory effect (IC50: 20 microM) on the activity of polyphosphoinositide-specific phospholipase C in purified plasma membranes. These results demonstrate that the plant phosphoinositide system can be activated by amphiphilic peptides in a manner analogous to that observed in specialized mammalian cells but that important functional components are altered, or lost, by the disruption of the intact cell state.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Daucus carota; Inositol 1,4,5-Trisphosphate; Intercellular Signaling Peptides and Proteins; Melitten; Peptides; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Wasp Venoms

The effects of various calmodulin inhibitors were examined on the Na/Ca exchange current in single cardiac ventricular cells of the guinea-pig using the whole-cell patch-clamp technique. External application of W-7 and trifluoperazine inhibited Na/Ca exchange current in a dose-dependent manner with IC50 values of 13 and 7 microM, respectively. W-5 inhibited the exchange current but less potently than W-7. More specific calmodulin inhibitors such as CGS 9343B and calmidazolium did not, however, decrease the current as significantly as expected. All these drugs inhibited the Na current more strongly than the Na/Ca exchange current. Ruthenium red (RR), another type of calmodulin inhibitor, did not decrease the exchange current by internal application. Neither mastoparan or melittin (calmodulin-binding peptides) inhibited the exchange current appreciably. RR and the peptides did not affect the Na current either. These results indicate that calmodulin may not involved in the activation of cardiac Na/Ca exchange or the Na current. Internal application of chymotrypsin inhibited the blocking effect of W-7 on the Na/Ca exchange current but not that on the Na current. These results indicate that W-7 blocks the Na/Ca exchange current not by binding to calmodulin but possibly by directly affecting an internal site of the exchanger itself and that the inhibitory action of W-7 is different on the Na/Ca exchange current and the Na current.

Topics: Animals; Benzimidazoles; Calcium; Calmodulin; Carrier Proteins; Guinea Pigs; Heart Ventricles; Imidazoles; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Melitten; Myocardium; Peptides; Ruthenium Red; Sodium; Sodium-Calcium Exchanger; Sulfonamides; Trifluoperazine; Wasp Venoms

The biologically relevant receptor for atrial natriuretic factor (ANF) has been shown to be membrane-bound guanylate cyclase. While guanylate cyclase is known to be activated by ANF and ATP, the molecular mechanism of the enzyme activation remains unclear. We now show that melittin, the main peptide toxin of bee venom, activates membrane-bound guanylate cyclase and potentiates ANF- and ATP-stimulated guanylate cyclase activity in rat lung membranes. Melittin stimulated basal guanylate cyclase activity by increasing the Vmax without significantly affecting the Km of the substrate, GTP. However, melittin enhances ANF- and ATP-stimulated enzyme activity by altering both the Vmax and the EC50 of ANF and ATP. Although melittin activates guanylate cyclase in crude membranes, it has little effect on the activity of the purified enzyme. The effect of melittin on guanylate cyclase activation in rat lung membranes is attenuated by the Ca2+ chelator, EGTA. These results suggest that the effects of melittin on guanylate cyclase activation may require the participation of accessory proteins or nonprotein factors. Therefore, melittin would be a valuable tool for exploring the molecular mechanisms of ANF-mediated guanylate cyclase activation.

Topics: Adenosine Triphosphate; Animals; Atrial Natriuretic Factor; Cell Membrane; Drug Synergism; Enzyme Activation; Guanylate Cyclase; Intercellular Signaling Peptides and Proteins; Kinetics; Lung; Male; Melitten; Peptides; Rats; Rats, Sprague-Dawley; Wasp Venoms

The interaction of heme with several amphiphilic peptides has been studied by absorption and fluorescence spectroscopy. The binding can be followed by the changes in the absorption spectrum of the heme group or by the decrease in the peptide tryptophan fluorescence due to energy transfer to the heme. Despite their small size, ranging from 26 residues for melittin to 14 for mastoporan, a high affinity for heme-CO and hemin-CN (Kd < 100 nM) may be observed. Spectral shifts in the absorption peaks and appreciable geminate recombination after photodissociation of CO from the complex peptide-heme-CO suggest the formation of a heme pocket, as for the natural heme proteins.. hemin-CN can be used as a probe for the interaction of calmodulin with these target peptides. Amphiphilic peptides such as melittin bind to calmodulin with a high (nM) affinity. While both the peptide and calcium-bound calmodulin bind heme-CO, only the peptide binds hemin-CN. These interactions permit studies of the competition between hemin-CN and calmodulin for binding to the peptide: while hemin-CN quenches the melittin tryptophan fluorescence, addition of calmodulin to the [melittin*hemin-CN] complex displaces the hemin-CN and the melittin tryptophan fluorescence is recovered.

Topics: Amino Acid Sequence; Binding Sites; Calmodulin; Carbon Dioxide; Heme; Hemin; Intercellular Signaling Peptides and Proteins; Melitten; Molecular Sequence Data; Peptides; Spectrophotometry; Wasp Venoms

We have examined the ability of two series of calcium binding site mutants of Drosophila calmodulin to form complexes with the model target peptides melittin and mastoparan. Unlike the wild-type complex, the 1:1 protein:melittin complexes formed by mutants of the C-terminal sites are unable to bind a second molecule of melittin. In contrast, a site 2 mutant shows increased ability to bind two molecules of melittin. For the shorter peptide mastoparan, most mutants form aberrant complexes that are best interpreted in terms of a model in which mastoparan interacts with both terminal domains of calmodulin. For two of the target enzymes of calmodulin, the three mutants which form mastoparan complexes most similar to the wild-type complex are also the best enzyme activators.

Topics: Binding Sites; Calcium; Calmodulin; Electrophoresis, Polyacrylamide Gel; Glycerol; Intercellular Signaling Peptides and Proteins; Melitten; Mutation; Peptides; Protein Conformation; Wasp Venoms

Two amphiphilic peptides from hymenopterid insects, melittin and mastoparan, stimulate secretion in a variety of cell types. In PC12 cells, both peptides stimulate calcium influx with melittin some 20-fold more potently than mastoparan. Melittin stimulates both breakdown of phosphoinositides (Pl) by phospholipase C to yield inositol phosphates and hydrolysis of phospholipids by phospholipase A2 to release arachidonic acid (AA). Mastoparan stimulates Pl breakdown, but has no effect on AA release. Maximal stimulation of Pl breakdown occurs at 1 to 2.5 micrograms/ml melittin and 30 micrograms/ml mastoparan, whereas maximal stimulation of AA release occurs at 2 to 5 micrograms/ml melittin. Organic calcium channel blockers (nifedipine, verapamil, diltiazem) have little or no effect on responses to the peptides. The influx of calcium elicited by melittin or mastoparan is completely or nearly completely blocked by inorganic calcium channel blockers (Co++, Mn++, Cd++). Mn++ and Cd++ inhibit melittin-induced Pl breakdown and AA release and mastoparan-induced Pl breakdown. Co++ has no effect on melittin-induced Pl breakdown and potentiates mastoparan-induced Pl breakdown. Pertussis toxin has no effect on the Pl breakdown induced by either peptide. The responses to melittin and mastoparan in PC12 cells are compared to those reported for maitotoxin.

Topics: Animals; Arachidonic Acid; Calcium; Calcium Channel Blockers; Intercellular Signaling Peptides and Proteins; Marine Toxins; Melitten; Oxocins; PC12 Cells; Peptides; Pertussis Toxin; Phosphatidylinositols; Phospholipases A; Phospholipases A2; Rats; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms

Topics: Animals; Gastric Mucosa; H(+)-K(+)-Exchanging ATPase; Intercellular Signaling Peptides and Proteins; Macromolecular Substances; Melitten; Peptides; Protein Structure, Secondary; Proton Pump Inhibitors; Swine; Wasp Venoms

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.

Topics: Amino Acid Sequence; Animals; Calcium; Cations, Divalent; Hydrogen-Ion Concentration; Intercellular Signaling Peptides and Proteins; Melitten; Molecular Sequence Data; Muscles; Peptides; Rabbits; Scattering, Radiation; Troponin; Troponin C; Wasp Venoms; X-Rays

The amphiphilic agents melittin, compound 48/80 and mastoparan inhibit ADP-ribosylation of porcine brain rho protein by Clostridium botulinum exoenzyme C3. However, ADP-ribosylation of recombinant rhoA expressed in E.coli was not inhibited by these agents. Accordingly, steady state GTP hydrolysis by recombinant rhoA was not stimulated by mastoparan, whereas GTP hydrolysis by porcine brain rho was stimulated 2.5-fold in the presence of this wasp venom. After microinjection of recombinant rhoA into Xenopus laevis oocytes the inhibitory effect of mastoparan on C3 ADP-ribosylation was restored. The data suggest that the amphiphilic agents tested are only active at the posttranslationally processed form of rho and that they exert their effects via the C-terminal end.

Topics: Adenosine Diphosphate Ribose; Animals; Autoradiography; Brain; Carbon Radioisotopes; Cloning, Molecular; Cytosol; Escherichia coli; GTP-Binding Proteins; Guanosine Triphosphate; Hemiterpenes; Intercellular Signaling Peptides and Proteins; Melitten; NAD; Oocytes; Organophosphorus Compounds; p-Methoxy-N-methylphenethylamine; Peptides; Protein Processing, Post-Translational; Recombinant Proteins; rhoA GTP-Binding Protein; Swine; Wasp Venoms; Xenopus laevis

In differentiated HL-60 cells the amphiphilic peptide mastoparan induces a dose-dependent stimulation of phosphoinositide breakdown with an EC50 value of 9 microM. Such stimulation can be markedly reduced by pretreatment of the cells with pertussis toxin (100 ng/ml, 2 h). In membranes obtained from differentiated HL-60 cells, guanine nucleotides stimulate the formation of IP2 and IP3. Calcium ions also induce phosphoinositide breakdown in this preparation independent of the presence of guanine nucleotides. In HL-60 cell membranes, mastoparan inhibited GTP gamma S-stimulation of phosphoinositide breakdown with an IC50 value of 3 microM. Such inhibitory activity of mastoparan also was present in membranes from cells pretreated with pertussis toxin. Calcium-induced stimulation of phosphoinositide breakdown was not significantly inhibited by mastoparan. The analogs mastoparan-X and polistes mastoparan had similar inhibitory activity, whereas the analog des-Ile1-Asn2-mastoparan was inactive. In permeabilized HL-60 cells mastoparan also inhibited phosphoinositide breakdown. Another amphiphilic peptide, melittin, was inactive in HL-60 intact cells, but similar to mastoparan, inhibited guanine nucleotide-induced phosphoinositide breakdown in HL-60 cell membranes and permeabilized cells. Thus, mastoparan peptides can stimulate phosphoinositide breakdown in intact HL-60 cells, probably through the interaction with a guanine nucleotide binding protein. In permeabilized cells and in cell membranes, mastoparan induces inhibition of guanine nucleotide-mediated phosphoinositide breakdown presumably through an interaction with an intracellular site. The inhibitory action of mastoparan and melittin is probably related to the amphiphilic character of these peptides.

Topics: Cell Membrane; Cell Membrane Permeability; Cell-Free System; Guanine Nucleotides; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Experimental; Melitten; Peptides; Phosphatidylinositols; Tumor Cells, Cultured; Type C Phospholipases; Wasp Venoms

Interactions of certain naturally occurring, amphiphilic polypeptides with membranes were investigated. Mastoparan (wasp venom toxin), melittin (bee venom toxin), cardiotoxin (cobra venom toxin), and polymyxin B (antibacterial antibiotic) inhibited protein kinase C stimulated by phosphatidylserine bilayer or arachidonate monomer and blocked binding of [3H] phorbol 12,13-dibutyrate to protein kinase C in the presence of phosphatidylserine bilayer, with IC50 values (concentrations causing 50% inhibition) of 1-8 microM. Mastoparan and polymyxin B were much less inhibitory (IC50, 10-20 microM), whereas melittin and cardiotoxin were similarly inhibitory (IC50, 1-4 microM), when protein kinase C was activated instead by synaptosomal membrane. Kinetic analysis indicate that mastoparan inhibited protein kinase C, assayed using phosphatidylserine or synaptosomal membrane as the phospholipid cofactor, competitively with the phospholipid cofactor, in a mixed manner with CaCl2 or diacylglycerol, noncompetitively with histone, and uncompetitively with ATP, with apparent Ki values of 1.6-18.7 microM. Inhibition of Na,K-ATPase in the membrane by these polypeptides had relative potencies different from those for their inhibition of protein kinase C activated by the same membrane preparation; mastoparan and melittin inhibited the two activities with comparable potencies, but polymyxin B and cardiotoxin were far less effective in inhibiting Na,K-ATPase. The same relative inhibitory potencies of the polypeptides (melittin greater than mastoparan greater than polymyxin B) for inhibition of Na,K-ATPase were also noted for their inhibition of Ca2+/calmodulin-dependent protein kinase II, 86Rb uptake (Na+ pump) by HL60 cells and the phorbol ester-induced differentiation of the leukemia cells. These findings were consistent with discrete interactions of the polypeptides with functionally distinct sites on the membrane, leading to differential inhibition of biological activities associated with the membrane. Actions of certain polypeptides appeared to be more specific compared to those of lipid second messengers such as lyso-phosphatidylcholine and sphingosine, and the antineoplastic ether lipid analogs such as 1-O-octadecyl-2-methyl-rac-glycero-3-ophosphocholine.

Topics: Animals; Biological Transport; Cell Differentiation; Cell Line; Cell Membrane; Cobra Cardiotoxin Proteins; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Melitten; Peptides; Polymyxin B; Protein Kinase Inhibitors; Sodium; Sodium-Potassium-Exchanging ATPase; Swine; Synaptosomes; Wasp Venoms

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.

Topics: Amino Acid Oxidoreductases; Animals; Calcineurin; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cells, Cultured; Cytosol; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; Imidazoles; Intercellular Signaling Peptides and Proteins; Kinetics; Melitten; Nitric Oxide; Nitric Oxide Synthase; Peptides; Phosphoprotein Phosphatases; Swine; Wasp Venoms

Topics: Cell Membrane; Cell Membrane Permeability; Hemolysis; Humans; Intercellular Signaling Peptides and Proteins; Mast Cells; Melitten; Peptides; Wasp Venoms

Arginine (AVP) and lysine vasopressin induce a weak but statistically significant increase in the water permeability of Amoeba proteus plasmalemma. Vasotocin and deaminovasopressin, which share the hydroosmotic properties of AVP on classical vertebrate systems, are without effects on Amoeba while SKF 101926, a synthetic AVP antagonist, is even more effective than the parent compound. Theophyllin and dibutyryl-cAMP do not affect AVP action on Amoeba. Lithium, oxytocin, and carbachol are also without effect. Thus, it is unlikely that either V2 (cAMP) or V1 (phosphatidylinositol choline) receptors are involved. A clear correlation has been found between the amphiphilic character of tested peptides and their effect on Amoeba water permeability. Classical amphiphilic peptides, melittin, mastoparan, and fragment 1-8 of alpha-neoendorphin, also increased water permeability in Amoeba. It is known that vasopressin can interact with artificial lipid membranes, increasing their permeability to water. We propose that amphiphilic members of the AVP family interact directly with the lipid phase of the Amoeba membrane. Their incorporation within the lipid bilayer may cause local disruptions or may create micellar water channels as shown for other amphiphilic proteins. Our observations provide a model for the early evolution of peptide hormone systems, preceding the appearance of specific membrane receptors and associated second messenger amplifying mechanisms.

Topics: Amoeba; Animals; Arginine Vasopressin; Cell Membrane; Cell Membrane Permeability; Deamino Arginine Vasopressin; Endorphins; Intercellular Signaling Peptides and Proteins; Lipid Metabolism; Lypressin; Melitten; Oxytocin; Peptides; Protein Precursors; Receptors, Angiotensin; Receptors, Vasopressin; Vasopressins; Wasp Venoms; Water

Crystals of troponin C are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).

Topics: Amino Acid Sequence; Animals; Calmodulin; Computer Simulation; Intercellular Signaling Peptides and Proteins; Melitten; Models, Molecular; Molecular Sequence Data; Peptides; Protein Conformation; Troponin; Troponin C; Turkeys; Wasp Venoms; Water; X-Ray Diffraction

Melittin stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect was rapid and associated with production and release of prostaglandin D2 and thromboxane B2. Indomethacin blocked the release of these eicosanoids and the stimulation of glycogenolysis induced by melittin. Ibuprofen blocked the release of prostaglandin D2 induced by melittin and markedly attenuated that of thromboxane B2. Interestingly, the initial burst of glucose output induced by melittin was not inhibited by ibuprofen, although the duration of the glycogenolytic action of the peptide was greatly diminished.

Topics: Animals; Bee Venoms; Female; Glycogen; Ibuprofen; Indomethacin; Intercellular Signaling Peptides and Proteins; Kinetics; Liver; Melitten; Peptides; Prostaglandin D2; Rats; Rats, Inbred Strains; Thromboxane B2; Vasoconstriction; Wasp Venoms

Calmodulin (CaM) fragments 1-77 (CaM 1-77) and 78-148 (CaM 78-148) were prepared by tryptic cleavage of CaM. CaM 78-148 exhibited Ca2+-dependent binding to mastoparan X, Polistes mastoparan, and melittin with apparent dissociation constants less than 0.2 microM as judged from changes in the fluorescence spectrum and anisotropy of the single tryptophan residue of each of these cationic, amphiphilic peptides. This interaction was accompanied by a large spectral blue shift of the peptide fluorescence spectrum. These findings are consistent with earlier results [Malencik, D.A., & Anderson, S.R. (1984) Biochemistry 23, 2420-2428] on the binding of mastoparan X to CaM fragment 72-148. The binding of the peptide to CaM 78-148 also caused a significant loss of the accessibility of the peptide tryptophan to the fluorescence quencher acrylamide. The CaM 78-148 induced effects on the fluorescence spectra and tryptophan accessibility of the peptides were most pronounced for mastoparan X, a peptide with tryptophan on the apolar face of the putative amphiphilic helix. The data were comparable with results from parallel experiments on the Ca2+-dependent interaction of these peptides with intact CaM. Difference circular dichroic spectra suggested that binding to CaM 78-148 was associated with the induction of considerable degrees of helicity in the amphiphilic peptides, which by themselves have predominantly random coil structures in aqueous solution. This finding is also reminiscent of the interaction of these peptides with intact CaM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Bee Venoms; Calmodulin; Intercellular Signaling Peptides and Proteins; Kinetics; Melitten; Peptide Fragments; Peptides; Protein Binding; Spectrometry, Fluorescence; Trypsin; Tryptophan; Wasp Venoms

Effects of phospholipase A2-activators, melittin and mastoparan, on rat anterior pituitary cells were studied by use of the electron microscope. Rat anterior pituitaries were incubated in HEPES buffer containing 20 micrograms/ml of melittin or the same dose of mastoparan for 5 min, 10 min and 20 min. Features indicating discharge of granule contents by exocytosis were increased with time, and the simultaneous extrusion of a number of secretory granules, named "multigranular exocytosis", was often recognized in addition to single-granule exocytosis at 10 min and 20 min. Most membrane pits, where the multigranular exocytosis as well as the single-granule exocytosis occurred, were coated. Moreover, a large number of vesicles coated or non-coated were distributed near the trans side of the Golgi apparatus of melittin-treated or mastoparan-treated cells after 20 min. These vesicles might be related to membrane internalized from the excess surface membrane derived from the limiting membrane of exocytosed granules. These observations indicate that phospholipase A2-activators induce hormone release involving membrane fusion between limiting membranes of secretory granules, and between granule-limiting membrane and plasma membrane in rat anterior pituitary cells.

Topics: Animals; Bee Venoms; Cell Membrane; Cytoplasmic Granules; Exocytosis; Golgi Apparatus; Intercellular Signaling Peptides and Proteins; Male; Melitten; Microscopy, Electron; Peptides; Phospholipases A; Phospholipases A2; Pituitary Gland, Anterior; Rats; Rats, Inbred Strains; Wasp Venoms

Observations on the intrinsic fluorescence of a high affinity calmodulin-binding peptide, Polistes mastoparan, reveal a spectroscopically distinct peptide complex present at maximum concentration when 2 mol Ca+2 are bound per mol calmodulin. The intermediate is detectable only in solutions where calcium is limiting. The results are consistent with cooperative binding of the first two equivalents of calcium by calmodulin.

Topics: Animals; Bee Venoms; Calcium; Calmodulin; Intercellular Signaling Peptides and Proteins; Melitten; Peptides; Spectrometry, Fluorescence; Swine; Tyrosine; Wasp Venoms

A wide variety of structurally different calmodulin antagonists enhanced the cytotoxicity of bleomycin A2 to leukemic L1210 cells. This potentiation occurred with nontoxic concentrations of calmodulin antagonists. The most potent blockers of L1210 calmodulin activity, melittin and mastoparan, were the most potent potentiators of bleomycin A2 cytotoxicity. Less potent agents such as pimozide, a diphenylbutylpiperidine, trifluoperazine and chlorpromazine, phenothiazines, and W-7, a naphthalene sulfonamide, required higher concentrations for potentiation of bleomycin A2-induced cytotoxicity, while homologs that lack anticalmodulin activity failed to increase the cytotoxicity seen with bleomycin A2. The potentiation of bleomycin A2 cytotoxicity was not due to an elevated cellular content of bleomycin A2 or to inhibition of bleomycin A2 inactivation. Using alkaline elution techniques, we found that pimozide increased bleomycin A2-induced DNA damage in intact L1210 cells. Pimozide did not, however, directly increase the formation of reactive species by bleomycin as measured by single or double strand breakage of covalently closed circular DNA. Thus, the potentiation of bleomycin cytotoxicity by these agents appears to be mediated by an increased damage to cellular DNA; this may be due to inhibition of DNA repair. The hypothesized calmodulin-dependent mechanism was not shared by all agents that caused breaks in DNA because no potentiation in cytotoxicity was observed when calmodulin antagonists were combined with either etoposide or X-irradiation.

Topics: Animals; Bleomycin; Calmodulin; Cell Survival; Chlorpromazine; DNA; Drug Synergism; Intercellular Signaling Peptides and Proteins; Leukemia L1210; Melitten; Mice; Peptides; Pimozide; Sulfonamides; Trifluoperazine; Wasp Venoms