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PubMed1994

Inositol(1,4,5)trisphosphate production in plant cells: stimulation by the venom peptides, melittin and mastoparan.

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.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

The influence of sterols on the sensitivity of lipid bilayers to melittin.

The sensitivity of planar lipid bilayers to the permeabalizing effect of melittin was evaluated when sterols of varying structure were incorporated into the membrane. The addition of increasing amount of cholesterol (0-50 mole %) decreased the sensitivity of membranes formed from negatively charged phospholipids to melittin but did not (in amount of up to 66 mole %) change the sensitivity of membranes formed from zwitterionic lipids. 7-Dehydrocholesterol, stigmasterol and ergosterol had the same ability as that of cholesterol to decrease the membrane sensitivity to melittin, while lanosterol had no effect on the sensitivity of membranes to melittin. The results suggest that the effect of sterols is complex and cannot be explained only by a direct interaction of melittin with cholesterol, by a decrease of membrane fluidity, or by changes in distribution of surface charge.

Cholesterol↗
PubMed1995

Such hydrophobic peptides as dansylated mastoparan can elevate the fertilization membrane of sea urchin eggs.

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.

Amino Acid Sequence↗
PubMed1996

Modulation of cell death pathways to apoptosis and necrosis of H2O2-treated rat thymocytes by lipocortin I.

Lipocortin I, also called annexin I, a calcium and phospholipid binding protein, protected rat thymocytes from H2O2-elicited necrosis and facilitated H2O2-induced apoptosis, while anti-lipocortin I antibody enhanced H202-elicited necrosis by blocking H202-induced apoptosis. Essentially similar results were obtained with phospholipase A2 inhibitors and activators such as 3,4-octyadecyl-benzylacrylic acid and melittin, respectively. Available evidence suggests that lipocortin I modulates signals for cell death pathways of H2O2-treated rat thymocytes to apoptosis and necrosis by regulating cellular phospholipase A2 activities but not by inhibiting membrane lipid peroxidation.

Animals↗
PubMed1997

Melittin binds to secretory phospholipase A2 and inhibits its enzymatic activity.

Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.

Animals↗
PubMed1998

Expression of antimicrobial peptides has an antitumour effect in human cells.

The antimicrobial peptides cecropin and melittin are known to exhibit antitumour activity in tumour derived cell lines. To achieve a similar effect in vivo these peptides would have to be given repeatedly to maintain therapeutic levels, which may be pharmacologically unfavourable. The expression of the genes encoding such antimicrobial peptides in the desired cell type may circumvent these problems. Expression constructs carrying cecropin or melittin have been introduced into a human bladder carcinoma derived cell line and the resultant cell clones analysed for tumorigenicity in nude mice. Expression of cecropin resulted in either a complete loss of tumorigenicity in some clones or reduced tumorigenicity, as measured by latency of tumour formation. These results suggest that vector mediated delivery of this gene to tumour cells may prove useful for cancer gene therapy.

Animals↗
PubMed1998

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells.

Phospholipase D (PLD) in lymphocytic mouse leukemic L1210 cells has been found to be activated by oleate both in vitro and in intact cells. The PLD activity was measured by phosphatidylethanol produced from radiolabeled phosphatidylcholine or myristic acid in the presence of ethanol. This oleate-activated PLD was further characterized in intact cells and compared with that in HL60 cells. Unlike PLD in HL60 cells, the PLD in L1210 cells was activated by unsaturated fatty acids, stimulated by melittin, insensitive to guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PIP2), independent of phorbol 12-myristate 13-acetate (PMA) and staurosporine, and inhibited by pervanadate. These observations indicate that the PLD present in L1210 cells is distinct from that in HL60 cells. Key PLD properties of L1210 cells such as insensitivity to GTP gamma S, ARF, PIP2, or PMA were in good agreement with currently known in vitro properties of the oleate-activated PLD found in mammalian sources. Therefore, the L1210 cells could be used as an intact-cell source for an oleate-activated PLD.

ADP-Ribosylation Factors↗
PubMed2000

The cyclic AMP-protein kinase A pathway restrains islet phospholipase A(2) activation.

Although phospholipase A(2) (PLA(2)) is of importance for insulin secretion, it is not established how it relates to other signalling mechanisms. This study examined the crosstalk between PLA(2) and the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in isolated rat islets. Forskolin, IBMX, and dbcAMP reduced [(3)H]arachidonic acid ([(3)H]AA) efflux from prelabelled islets during PLA(2) activation by mellitin or cholecystokinin (CCK-8), while efflux induced by carbachol was unaffected. The PKA inhibitor myrPKI(14-22) prevented this reduction of CCK-8-induced efflux. Glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and vasoactive intestinal polypeptide (VIP) diminished CCK-8-induced efflux. Also in the absence of Ca(2+), forskolin/IBMX and dbcAMP reduced CCK-8-induced efflux. In parallel with effects on [(3)H]AA, the expected additive insulin secretion induced by mellitin or CCK-8 in combination with forskolin or GLP-1, respectively, was reduced. In conclusion, the cAMP-PKA pathway restrains both Ca(2+)-dependent and Ca(2+)-independent PLA(2) activation, indicating a regulating crosstalk between these two pathways.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Islet phospholipase A(2) activation is potentiated in insulin resistant mice.

Insulin resistance is followed by an islet adaptation resulting in a compensating increase in insulin secretion and hyperinsulinemia. The mechanism underlying this increased insulin secretion is not established. We studied whether islet phospholipase A(2) (PLA(2)) contributes by using C57BL/6J mice fed a high-fat diet, since we previously showed that the insulin responses to the two PLA(2)-activating insulin secretagogues carbachol and cholecystokinin (CCK) are enhanced in this model. CCK (100 nM) and carbachol (100 microM) stimulated [(3)H]AA efflux, reflecting PLA(2) activation, both in islets from mice after 12 weeks on high-fat diet and in controls. The efflux increase was more pronounced in islets from high-fat diet-fed mice during both CCK (by 93 +/- 46%; P = 0. 034) and carbachol (by 64 +/- 22%; P = 0.009) stimulation. Also a direct PLA(2) activation by mellitin (2 microg/ml) elicited a potentiated efflux in islets from the insulin-resistant mice (by 361 +/- 107%; P = 0.002). The results suggest that exaggerated non-glucose-induced PLA(2) activation contributes to the islet compensation in insulin resistance.

Animals↗
PubMed2002

Melittin-GM1 interaction: a model for a side-by-side complex.

We report here the interaction of melittin with ganglioside GM1 by steady-state fluorescence, one-dimensional (1)H NMR spectroscopy and molecular modeling. In the presence of GM1 the emission maximum of melittin is blue shifted and fluorescence quenching efficiencies of iodide and acrylamide are substantially reduced, indicating a shielding of tryptophan of melittin from aqueous environment. Significant line broadening of NMR resonances of melittin, suggestive of motional restriction, is observed. Molecular modeling indicates a melittin-GM1 complex with N-terminal hydrophobic stretch of melittin associating with the ceramide tail and C-terminal hydrophilic end of melittin having favorable electrostatic interaction with the carbohydrate head group of GM1.

Computer Simulation↗
PubMed2002

Effects of a lytic peptide conjugated to beta HCG on ovarian cancer: studies in vitro and in vivo.

OBJECTIVE: The aim of this study was to determine the in vitro and in vivo effects of the lytic peptide, hecate, alone and conjugated to a 15-amino-acid fragment of the beta-chain of hCG (hecate-beta hCG) on the ovarian carcinoma cell line NIH: OVCAR-3 and determine the expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors in cell cultures and tumor tissues. METHODS: For in vitro studies, hecate or hecate-beta hCG was added to cultures of ovarian cancer cells in the presence or absence of estradiol or follicle stimulating hormone. The cytotoxicity of lytic peptides was measured by trypan blue exclusion and lactate dehydrogenase release. For in vivo studies, OVCAR-3 xenografts were established in female athymic nude mice which were then treated once per week for 3 weeks with hecate or hecate-beta hCG via the lateral tail vein. An immunohistochemical method was used to analyze the expression of LH/hCG receptor in tumor and culture cells. RESULTS: In in vitro studies, both hecate-beta hCG and hecate destroyed ovarian cancer cells (NIH: OVCAR-3) in a dose-dependent manner. Removal of steroids from the culture medium reduced the sensitivity of the OVCAR-3 cell line to the hecate-beta hCG in a reversible manner. In in vivo studies, the average tumor volume and tumor burden in lytic peptide treated animals were reduced. In the groups of animals treated by hecate, hecate-beta hCG, and estradiol + hecate-beta hCG, tumor volumes after treatment expressed as a percentage of increase (197.4 +/- 21.72, 199.0 +/- 18.57, and 193.8 +/- 22.94%, respectively) were reduced, compared to control (263.0 +/- 21.72%) animals (P < 0.05). Immunocytochemical studies revealed the expression of LH/hCG receptor protein in the OVCAR-3 cells and tumor tissues. CONCLUSION: Hecate-beta hCG is a putative candidate for treating ovarian cancer.

Animals↗
PubMed1994

Structure and orientation of the pore-forming peptide, melittin, in lipid bilayers.

Ten analogues of the 26-residue, bee venom peptide, melittin (H3N(+)-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2), were synthesized, each with 13C enrichment of a single peptide carbonyl carbon. These peptides were incorporated into bilayers of the diether lipid, ditetradecylphosphatidylcholine, aligned between stacked glass plates. Solid-state 13C nuclear magnetic resonance spectra were obtained as a function of the angle between the bilayer planes and the magnetic field of the spectrometers, and at temperatures above and below the lipid gel-to-liquid crystalline transition temperature, Tc. For bilayers aligned with the normal along the applied magnetic field there was no shift in the carbonyl resonances of residues Ile2, Ala4, Leu9, Leu13, or Ala15, with minor changes for residues Val8 and Ile20, and small changes at Val5, Leu6 and Ile17 on immobilization of the peptide below Tc. In contrast, the spectra for bilayers aligned at right angles to the field showed greatly increased anisotropy below Tc for all analogues. From these experiments it was evident that the peptide was well-aligned in the bilayers and reoriented about the bilayer normal. The observed reduced chemical shift anisotropies and the chemical shifts were consistent with melittin adopting a helical conformation with a transbilayer orientation in the lipid membranes. With the exception of Ile17, there was no apparent difference between the behaviour of residues in the two segments that form separate helices in the water-soluble form of the peptide, suggesting that in membranes the angle between the helices is greater than the 120 degrees observed in the crystal form.

Amino Acid Sequence↗
PubMed1998

Group additive contributions to the alcohol-induced alpha-helix formation of melittin: implication for the mechanism of the alcohol effects on proteins.

Detailed analysis of the effects of alcohols on proteins and peptides is important because of its wide range of applications in many fields. Whereas melittin, a major component of honeybee venom, is unfolded in an aqueous environment, the addition of alcohols induces an alpha-helical structure. We used circular dichroism (CD) to compare the effects of various alcohols on melittin. Whereas the alpha-helical state was basically independent of alcohol species, the effectiveness of alcohols varied significantly. Among alkanols, the effectiveness was proportional to the bulkiness of hydrocarbon groups, indicating that the hydrocarbon group contributes positively to the alcohol effects. Comparison of alcohols with the same hydrocarbon group but a different number of hydroxyl groups showed that the hydroxyl groups contribute negatively to the alcohol effects. Comparison of several halogenols indicated that halogen increases the effectiveness in the order of F < Cl < Br. These results suggested that the effects of alcohol can be interpreted by the additive contributions of each of the constituent groups of the alcohol, which are proportional to the solvent-accessible surface area. However, for markedly effective alcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol, the aggregation of alcohols was suggested to further enhance these effects. We have constructed equations for estimating the effectiveness of alcohols in inducing alpha-helical structure, which should also be useful for predicting the other effects of alcohols on proteins.

Alcohols↗
PubMed1998

Topology of the calmodulin-melittin complex.

The topology of the Ca2+-calmodulin-melittin ternary complex has been investigated by a combined strategy which integrates limited proteolysis and cross-linking experiments with mass spectrometric methodologies. The rationale behind the methods is that the interface regions of two interacting proteins are accessible to the solvent in the isolated molecules, whereas they become protected following the formation of the complex. Therefore, when limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential peptide maps are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly controlled conditions lead to the identification of spatially closed amino acid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify covalently linked residues. Our results show that melittin interacts with calmodulin by adopting a parallel orientation, i.e. the N and C-terminal halves of the peptide are anchored to the amino and carboxy-terminal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model of the complex was designed and refined on the basis of the experimental results, supporting the above conclusions. This finding reveals a further dimension to the already remarkable capability of calmodulin in binding different protein substrates, providing this protein with the capability of regulating an even larger number of enzymes.

Amino Acid Sequence↗
PubMed1999

Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin.

Membranes have a potent ability to promote secondary structure formation in a wide range of membrane-active peptides, believed to be due to a reduction through hydrogen bonding of the energetic cost of partitioning peptide bonds. This process is of fundamental importance for understanding the mechanism of action of toxins and antimicrobial peptides and the stability of membrane proteins. A classic example of membrane-induced folding is the bee-venom peptide melittin that is largely unstructured when free in solution, but strongly adopts an amphipathic alpha-helical conformation when partitioned into membranes. We have determined the energetics of melittin helix formation through measurements of the partitioning free energies and the helicities of native melittin and of a diastereomeric analog with four d-amino acids (d4,l-melittin). Because D4,l-melittin has little secondary structure in either the free or bound forms, it serves as a model for the experimentally inaccessible unfolded bound form of native melittin. The partitioning of native melittin into large unilamellar phosphocholine vesicles is 5.0(+/-0.7) kcal mol-1 more favorable than the partitioning of d4,l-melittin (1 cal=4.186 J). Differences in the circular dichroism spectra of the two forms of melittin indicate that bound native melittin is more helical than bound d4, l-melittin by about 12 residues. These findings disclose that the free energy reduction per residue accompanying the folding of melittin in membrane interfaces is about 0.4 kcal mol-1, consistent with the hypothesis that hydrogen bonding reduces the high cost of partitioning peptide bonds. A value of 0.6 kcal mol-1 per residue has been observed for beta-sheet formation by a hexapeptide model system. These two values provide a useful rule of thumb for estimating the energetic consequences of membrane-induced secondary structure formation.

Amino Acid Sequence↗
PubMed1998

Phospholipase A2-mediated activation of phospholipase D in rat heart sarcolemma.

The effect of phospholipase A2 (PLA2)-dependent release of unsaturated fatty acids (FA) on phospholipase D (PLD) function was examined in purified sarcolemmal (SL) membranes isolated from rat heart. PLD hydrolytic activity was determined by measuring either [14C] phosphatidic acid formation from exogenous [14C] phosphatidylcholine (PtdCho) or [3H] choline release from prelabelled SL Ptd[3H]choline. SL membranes with endogenous [3H] PtdCho that were prelabelled with [3H] myristic acid were used for testing PLD transphosphatidylation activity. Exogenous cis-unsaturated FA, arachidonate and oleate, significantly enhanced the [3H] choline formation at 50 and 100 microM, respectively; their effect was maximal at 250 microM and declined at higher concentrations. Use of melittin (which stimulates membrane-bound PLA2, thus releasing FA) or exogenous PLA2 reproduced the stimulatory effect of added arachidonate and oleate. Under melittin, PLA2-dependent FA release was strongly correlated (r = 0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphatidylation activity confirmed the augmented catalytic rate of PLD by these agents. Melittin-evoked PLD activation was completely blocked by 1 microM E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective inhibitor of Ca(2+)-independent v Ca(2-)-dependent PLA2, thus indicating that PLD stimulation under melittin occurred via PLA2. Activity measurement and Western blotting studies revealed the presence of a Ca(2+)-independent, high molecular weight (110 kDa) PLA2 in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These results suggest that Ca(2+)-independent PLA2 and subsequent endogenous mobilization of sn-2 unsaturated FA modulate PLD activity in heart SL membranes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA2 as well as in heart diseases characterized by PLA2 pathological dysfunction.

Animals↗
PubMed1997

PhoP/Q regulated genes in Salmonella typhi identification of melittin sensitive mutants.

Many of the genes (pags (phoP activated genes) and prgs (phoP repressed genes)) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival of Salmonella typhimurium in murine macrophages and pathogenesis in mice. Although a great deal is known about the S. typhimurium phoP/Q regulon, little has been done with the human specific pathogen S. typhi, prompting us to investigate S. typhi phoP/Q regulated genes. Isogenic phoP12 (null) and phoP24 (constitutive) strains were constructed in S. typhi Ty2 and S. typhimurium C5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested that S. typhi and S. typhimurium may have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of a phoP12 mutant, creating lacZ-gene fusions, and screening for Lac+ or Lac- colonies. A mobilizable plasmid carrying the phoP24 mutant gene was conjugated into these insertion mutants. Those that changed from Lac- to Lac+ were inferred to be pag::MudJ insertions and those that changed from Lac+ to Lac- were inferred to be prg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termed pqa (PhoPQ activated) and pqr (PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. The pqa/pqr::MudJ mutations were transduced into S. typhi phoP+ and phoP24 strains by Vi-l phage transduction. Characterization of the mutants (Southern blot analysis, beta-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.

Animals↗
PubMed1995

Conversion of a bovine UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase to a soluble, secreted enzyme, and expression in Sf9 cells.

A soluble, secreted UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase, was prepared by substituting the honeybee melittin leader sequence for the sequences coding for the cytoplasmic and membrane spanning domains of a cloned, bovine full-length cDNA (F. L. Homa et al., 1993, J. Biol. Chem. 268, 12609-12616). When this construct was expressed in insect cells using a recombinant baculovirus, a fully active soluble enzyme was recovered from the culture medium. In large-scale preparations, approximately 2-3 mg of enzyme protein was produced per liter of medium. A one-step purification of the soluble molecule on apomucin-Sepharose yielded an essentially homogeneous enzyme preparation. The purified soluble enzyme has a molecular mass of approximately 61 kDa and appears to contain both N- an O-linked oligosaccharides. NH2-terminal sequencing demonstrated that the melittin leader sequence is cleaved at the predicted site and amino acid analysis gave results in close agreement with the composition predicted by the nucleic acid sequence. The enzymatic properties of the soluble, recombinant molecule are similar to those of the enzymes isolated from bovine colostrum and porcine submaxillary gland: the specific activity is approximately 2160 U/mg protein and the Kms for UDP-GalNAc and the synthetic acceptor peptides PPASTSAPG and PPDAASAAPLR are approximately 1.7 microM, 6.5 mM, and 3.6 mM, respectively.

Amino Acid Sequence↗