[Cardiotropic action of bee venom and melitten].
Explore the source record for details and available documents.
Animals↗
SEARCH · Search PubMed
Results for “Melitten”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
[Effect of melitten from bee venom on mitochondrial functions].
Effect of different concentrations of bee venom melittin on respiration, mitochondrial oxidative phosphorylation, and permeability of mitochondrial inner membranes for different ions is studied. Low melittin concentrations (1-2 mcM) increased in several times the rate of H+, K+, Na+ ions and Tris transport, in a lesser degree--bivalent ions and Cl- transport and did not affect the permeability for succrose. Thus, the uncoupling effect of low melittin concentrations on mitochondrial energetic apparatus is due to unspecific increase of inner membrane permeability. High melittin concentrations (50-100 mcM) inhibited mitochondrial respiration due to disturbances of membrane and organized respication complexes structures.
Bee Venoms↗
The linker of calmodulin lacking Glu84 is elongated in solution, although it is bent in the crystal.
The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 A and 62.5 A, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 A and 55.0 A, and smaller than those expected from the crystal structure of wild type, 22.8 A and 67.5 A. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether.
Calcium↗
Stability of secondary structural elements in a solvent-free environment: the alpha helix.
The stability of the alpha helix as an element of secondary structure is examined in the absence of solvation, in the gas phase. Mass-analyzed ion kinetic energy (MIKE) spectrometry was applied to measure intercharge repulsion and intercharge distance in multiply protonated melittin, a polypeptide known to possess a stable helical structure in a number of different environments. The experimental results, interpreted in combination with molecular mechanics calculations, suggest that triply charged melittin retains its secondary structure in the gas phase. The stability if the alpha-helical conformation of the polypeptide in the absence of solvent molecules reflects the fact that a network of intrinsic helical hydrogen bonds is energetically more favorable than unfolded conformations.
Melitten↗
Integrated multichannel microchip electrospray ionization mass spectrometry: analysis of peptides from on-chip tryptic digestion of melittin.
In continuation of our work to develop an integrated multichannel microchip interfaced to electrospray mass spectrometry (ESI-MS), this paper demonstrates one of several applications of this approach in monitoring tryptic digestion products. The multichannel microchip allowed integration of sample preparation onto the microchip to facilitate the analysis process. Melittin was selected as a model oligopeptide because it possesses a cluster of four adjacent basic residues which enable probing the site specificity of trypsin as a function of digest times. Reactions were performed on-chip in different wells for specific time periods and then analyzed by infusion from the microchip by ESI-MS, using leucine enkephalin as internal standard. The rate of formation and disappearance of the molecular ion and individual fragments was followed for a melittin to trypsin concentration ratio of 300:1. The results indicate the potential of integrating enzymatic reactions with multichannel microchip ESI-MS for automated optimization of reaction condition while consuming only small amounts of sample.
Amino Acid Sequence↗
Magnetic sector-ion trap mass spectrometry with electrospray ionization for high sensitivity peptide sequencing.
A hybrid mass spectrometer composed of a high resolution double focusing instrument (electrostatic analyzer-magnetic sector, EB) and an ion trap analyzer (T) exhibits high sensitivity performance for peptide sequencing with electrospray ionization (ESI). MS2 and MS3 experiments for multiply charged tryptic peptides and larger peptides (e.g., melittin, 2.8 kDa) generate sequence-informative product ions. Collisionally activated dissociation (CAD) of selected precursor ions can also be performed in the interface between the double focusing analyzer and the ion trap (transfer octapole region) to generate product ions. With a low-flow micro-ESI source, which can deliver analyte solution to the source at a flowrate of 10-200 nL/min, tandem mass spectra can be obtained from sub-fmol amounts of melittin. The high resolving power of the MS-I stage combined with the efficiency of the ion trap stage allows for high resolution precursor ion selection with subsequent highly sensitive tandem mass spectrometry (MS/MS) analysis.
Acyl Carrier Protein↗
Binding of basic amphipathic peptides to neutral phospholipid membranes: a thermodynamic study applied to dansyl-labeled melittin and substance P analogues.
A thermodynamic approach is proposed to quantitatively analyze the binding isotherms of peptides to model membranes as a function of one adjustable parameter, the actual peptide charge in solution z(p)+. The main features of this approach are a theoretical expression for the partition coefficient calculated from the molar free energies of the peptide in the aqueous and lipid phases, an equation proposed by S. Stankowski [(1991) Biophysical Journal, Vol. 60, p. 341] to evaluate the activity coefficient of the peptide in the lipid phase, and the Debye-Hückel equation that quantifies the activity coefficient of the peptide in the aqueous phase. To assess the validity of this approach we have studied, by means of steady-state fluorescence spectroscopy, the interaction of basic amphipathic peptides such as melittin and its dansylcadaverine analogue (DNC-melittin), as well as a new fluorescent analogue of substance P, SP (DNC-SP) with neutral phospholipid membranes. A consistent quantitative analysis of each binding curve was achieved. The z(p)+ values obtained were always found to be lower than the physical charge of the peptide. These z(p)+ values can be rationalized by considering that the peptide charged groups are strongly associated with counterions in buffer solution at a given ionic strength. The partition coefficients theoretically derived using the z(p)+ values were in agreement with those deduced from the Gouy-Chapman formalism. Ultimately, from the z(p)+ values the molar free energies for the free and lipid-bound states of the peptides have been calculated.
Amino Acid Sequence↗
Oligomerization of smooth muscle myosin light chain kinase and its modifications by melittin and calmodulin.
The catalytic activity of smooth muscle myosin light chain kinase (MLCKase) requires the presence of calcium and calmodulin [CM; J. T. Stull et al. (1993) Molecular and Cellular Biochemistry, Vols. 127/128, pp. 229-237] and can also be modified through its own oligomerization [E. B. Babiychuk et al. (1995) Biochemistry, Vol. 34, pp. 6366-6372]. In the present report we demonstrate that melittin, one of the most potent CM antagonists, interacted reversibly with the MLCKase apoenzyme with affinities comparable to those of CM and influenced the oligomeric state of the kinase. At low melittin to kinase ratios the kinase formed insoluble oligomers (aggregates) while at higher melittin concentrations it existed predominantly as soluble oligomers revealed by cross-linking as octamers and hexamers. The kinase alone exhibited similar biphasic solubility within a 5-30 microM range and its solubility was strongly influenced by the ionic strength of the medium. Melittin was also found to promote both the aggregation of the purified 24-kDa C-terminal fragment of the kinase and its analogue telokin, as well as of myosin light chains, but had no effect on the solubility of bovine serum albumin, caldesmon, or calmodulin. These data and our cross-linkage experiments indicate that the insoluble kinase oligomers arose via melittin-induced aggregation of the kinase dimers in which the relokin-like domain played a main role. The soluble oligomers, in turn, were formed after saturation of the kinase with melittin, which resulted in a weakening of the interaction between the protomers with an increase of the long-range order within the oligomers. This interpretation is consistent with the observed effects of melittin on MLCKase catalytic and autocatalytic activities. At concentrations of melittin required to produce soluble oligomers, the binding of the kinase to myosin filaments was considerably enhanced. A plausible mechanism for the formation of the soluble oligomers and aggregates is suggested and its relation to the possible MLCKase assemblies discussed in terms of a model.
Animals↗
Conformational analysis of the 20-residue membrane-bound portion of melittin by conformational space annealing.
The conformational space of the 20-residue membrane-bound portion of melittin has been investigated extensively with the conformational space annealing (CSA) method and the ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm. Starting from random conformations, the CSA method finds that there are at least five different classes of conformations, within 4 kcal/mol, which have distinct backbone structures. We find that the lowest energy conformation of this peptide from previous investigations is not the global minimum-energy conformation (GMEC); but it belongs to the second lowest energy class of the five classes found here. In four independent runs, one conformation is found repeatedly as the lowest energy conformation of the peptide (two of the four lowest energy conformations are identical; the other two have essentially identical backbone conformations but slightly different side-chain conformations). We propose this conformation, whose energy is lower than that found previously by 1.9 kcal/mol, as the GMEC of the ECEPP/3 force field. The structure of the proposed GMEC is less helical and more compact than the previous one. It appears that the CSA method can find several classes of conformations of a 20-residue peptide starting from random conformations utilizing only its amino acid sequence information. The proposed GMEC has also been found with a modified electrostatically driven Monte Carlo method [D. R. Ripoll, A. Liwo, and H. A. Scheraga (1998) "New Developments of the Electrostatically Driven Monte Carlo Method: Test on the Membrane-Bound Portion of Melittin," Biopolymers, Vol. 46, pp. 117-126].
Algorithms↗
New developments of the electrostatically driven Monte Carlo method: test on the membrane-bound portion of melittin.
The electrostatically driven Monte Carlo (EDMC) method has been greatly improved by adding a series of new features, including a procedure for cluster analysis of the accepted conformations. This information is used to guide the search for the global energy minimum. Alternative procedures for generating perturbed conformations to sample the conformational space were also included. These procedures enhance the efficiency of the method by generating a larger number of low-energy conformations. The improved EDMC method has been used to explore the conformational space of a 20-residue polypeptide chain whose sequence corresponds to the membrane-bound portion of melittin. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm was used to describe the conformational energy of the chain. After an exhaustive search involving 14 independent runs, the lowest energy conformation (LEC) (-91.0 kcal/mol) of the entire study was encountered in four of the runs, while conformations higher in energy by no more than 1.8 kcal/mol were found in the remaining runs with the exception of one of them (run 8). The LEC is identical to the conformation found recently by J. Lee, H. A. Scheraga, and S. Rackovsky [(1988) "Conformational Analysis of the 20-Residue Membrane-Bound Portion of Melittin by Conformational Space Annealing," Biopolymers, Vol. 46, pp. 103-115] as the lowest energy conformation obtained in their study using the conformational space annealing method. These results suggest that this conformation corresponds to the global energy minimum of the ECEPP/3 potential function for this specific sequence: it also appears to be the conformation of lowest free energy.
Amino Acid Sequence↗
Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.
Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.
Allergens↗
Phospholipase A2 activity is required for regeneration of sensory axons in cultured adult sciatic nerves.
The adult frog dorsal root ganglia (DRGs) and their sciatic nerves (ScN) survive in organ culture for several days. About 3 days after a local test crush, the sensory axons start to regenerate into the distal nerve stump at a rate of approximately 0.6-0.9 mm/day. The axonal outgrowth is inhibited in a non-toxic way by low concentrations of three different phospholipase A2 (PLA2) inhibitors: 4-bromophenacyl bromide (BPB), aristolochic acid, and oleyl-oxyethyl-phosphoryl-choline (OOPC). In contrast, the outgrowth was slightly stimulated by 0.2 microM melittin, a PLA2 activator. Most experiments refer to the effects of BPB, which was shown to almost completely inhibit outgrowth at a concentration which did not affect either ganglionic protein synthesis or axonal transport. Using a compartmental system it could clearly be shown that BPB exerted its action in the outgrowth region. Other experiments showed that the initial period (about 3 days), which precedes the outgrowth, was unaffected by BPB. Several structures, including axonal ones, showed immunoreactivity for the low molecular form of PLA2 (sPLA2). The results suggest that PLA2 activity plays an important role in nerve regeneration and exerts its action at a local level, where the growth cones move forward.
Acetophenones↗
Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: function of arachidonic acid.
Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 microM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 microM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A2, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4 alpha-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells.
3T3 Cells↗
Enhancement of stress-induced synthesis of hsp27 and alpha B crystallin by modulators of the arachidonic acid cascade.
The regulation by intrinsic factors of responses to stress of two small stress proteins, hsp27 and alpha B crystallin, was examined in C6 rat glioma cells. Levels of hsp27 and alpha B crystallin were low in C6 glioma cells in confluent cultures. However, levels of the two proteins increased after exposure of cells to heat (42 degrees C for 30 min) or arsenite (50 microM for 1 h) stress. When cells were exposed to arsenite or hear in the presence of indomethacin (50 microM), an inhibitor of cyclooxygenase, or in the presence of nordihydroguaiaretic acid (NDGA; 50 microM), an inhibitor of lipoxygenase, induction of hsp27 and alpha B crystallin was markedly stimulated as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. The presence of melittin (1 microM), an activator of phospholipase A2, during the stress period also stimulated the induction of the two proteins. The expression of hsp70 to each stress was also enhanced in the presence of indomethacin, NDGA, or melittin. The gel mobility shift assay revealed that these chemicals prolonged the arsenite-induced activation of heat shock element (HSE)-binding activity of heat shock transcriptional factor (HSF) in cells. Induction of hsp27 and alpha B crystallin in adrenal glands of heat-stressed (42 degrees C for 15 min) rats was also enhanced by prior injection of aspirin, another inhibitor of cyclooxygenase. These results indicate that the responses to stress of hsp27 and alpha B crystallin, as well as the response of hsp70, are coupled with the metabolic activity of the arachidonic acid cascade and the mechanism for regulation of stress responses observed in C6 cells is operative in tissues and organs in vivo.
Animals↗
Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation.
Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.
Adenosine Triphosphate↗
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.
24,25-Dihydroxyvitamin D 3↗
All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries.
The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D-amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.
Amino Acid Sequence↗
Molecular parameters in melittin immunogenicity.
Based on immunogenicity studies, two T-cell epitopes in melittin were found to be functional in guinea pigs, one being centrally located, the other one residing in the C-terminal chain. In Balb/c mice only the central epitope was found to be active. A human T-cell clone was found by T-cell proliferation studies to employ strictly the C-terminal chain. Truncation of melittin peptides at the N-terminus did not markedly affect the capacity of guinea pigs to develop anti-IgG responses towards peptidic epitopes and towards a C-terminally attached haptenic group. Attachment of various substituents inside and outside the T-cell epitopic areas had no marked effect on antibody responses. In contrast, the substituents positioned within a T-cell epitope abolished T-cell proliferation. This difference between whole animal data and cellular in vitro responses is presently not understood.
Amino Acid Sequence↗