Christa Mollay

Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

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

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

Enhancement of bee venom phospholipase A2 activity by melittin, direct lytic factor from cobra venom and polymyxin B☆

The peptide melittin and the enzyme phospholipase AZ, two main constituents of bee venom, show a marked synergism in their action on cell membranes. Under conditions where neither of the two substances has any deleterious effect, their combined action readily leads to the lysis of e.g. erythrocytes [l] . We have recently observed that mehttin forms a complex with lecithins provided the lipid is in the liquid-crystalline state [2j. This hydrophobic binding of melittin to phospholipids may be an intermediate step in the action of bee venom on cell membranes and thus be of relevance for explaining the synergistic action of venom components. If this argument is correct, one would expect that melittin stimulates the action of phospholipase only when complex formation between the peptide and the phospholipid can occur. In this report we present our results on the action of purified phospholipase AZ from bee venom on lecithin liposomes in the presence and absence of melittin. It could be shown that melittin stimulates the enzymatic hydrolysis of egg lecithin up to about 5-fold, while similar effects could not be observed with dipalmitoyllecithin. Previous experiments have shown that at the assay temperature melittin interacts with the former but not with the latter type of phospholipid. Starting from these findings, we have tested additional peptides known to interact with membranes: these

Fluorometric measurements on the interactton of melittin with lecithin

Abstract The interaction of melittin, a lytic peptide found in bee venom, with lecithins was studied. The single tryptophan residue present in melittin served as a fluorescence probe. Melittin and lecithins do form a complex, whereby the maximal emission wave-length shifts from 350 to 330 nm. This indicates a transfer of the indole ring from water to a hydrophobic environment. The melittin-lecithin complex can be isolated by chromatography on Sephadex G-200. This interaction is dependent on the concentration of lecithin, the ratio of peptide to lipid, and the fluidity of the micelles. Using again fluorescence measurements, no interaction between melittin and cholesterol could be detected. Lysolecithin, the product of the phospholipase A2 reaction, does form a complex with melittin, from which it can apparently be displaced by lecithin. A possible mechanism for the synergism of melittin and phospholipase A2 is discussed.

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm-1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm-1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5 degrees C for the pure lipid, the lower transition, exhibiting a 2 degree C width, is centered at 17 degrees C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7 degree C interval, occurs at approx. 29 degrees C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30 degrees C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.

Effect of melittin and melittin fragments on the thermotropic phase transition of dipalmitoyllecithin and on the amount of lipid-bound water

Melittin is the main cytolytic component of bee venom [l] . The first twenty amino acids of this peptide are mostly apolar whereas the C-terminal hexapeptide is polar and highly basic. The biological activity of melittin has been studied by biochemical and biophysical methods [2-91. Changes in the physical state of membrane phospholipids have been postulated to cause the biological effects of melittin such as increased susceptibility of lipids towards phospholipases AZ [3,7,8] , and at higher peptide concentrations, complete lysis of cells [2]. Melittin interacts with phospholipids both in liposomes [6] and in biomembranes [6,8] . According to recent evidence, this interaction causes a decrease in the mobility of the fatty acid chains and a rearrangement of the headgroup of phosphatides [S] . The hydrophobic segment of melittin, corresponding to the first nineteen residues (designated mell__rr,) and the basic heptapeptide (melae-s6) can be isolated. The effects of melittin and of these characteristic fragments on the gel to liquid crystalline phasetransition of dipalmitoylphosphatidylcholine and the amount of lipid bound water are described in this paper.

Detection and partial characterization of an amidating enzyme in skin secretion of Xenopus laevis

Peptidyl‐glycine‐α‐amidating monooxygenase has been purified about 150‐fold from skin secretion of Xenopus laevis by a three‐step procedure. The enzyme converts [14C]succinyl‐Ala‐Phe‐Gly to [14C]succinyl‐Ala‐Phe‐amide at both pH 5.5 and 8.4 in the presence of different metal ions and ascorbate or tetrahydropterin. In contrast to the mammalian enzyme, higher activities were detected at pH 5.5.

PREPROMELITTIN: SPECIFIC CLEAVAGE OF THE PRE- AND THE PROPEPTIDE IN VITRO

The venom gland of honeybees produces an aqueous secretion that contains as its main constituent the lytic peptide melittin.’ This peptide is derived from a precursor-promelittin-which has been detected in venom glands of bees previously fed with radioactive amino acids.’ The mRNA for melittin, as isolated and purified from venom glands of newly emerged queen bees,’,‘ yields in cell-free systems the translation product prepr~melit t in,~.~ a polypeptide of 70 amino acids, the smallest of the preproteins known to date. From a functional point of view, four regions can be discerned in the primary structure of prepromelittin (see FIGURE 1). Starting from the initiating methionine, these are the prepeptide (21 residues), the proregion (22 amino acids), melittin which is composed of 26 residues, and finally an additional glycine at the carboxyl end. A preprotein of such small size raises several questions about the detailed mechanism of the vectorial discharge of nascent polypeptide chains into the cisternae of the endoplasmic reticulum. If cleavage of the presequence in the case of prepromelittin occurs at or prior to the time of chain completion, the resulting peptide composed of 49 amino acids could then be barely protruding, at most by only a few residues, from the luminal side of the microsomal membrane, and its major part would still be buried in the large subunit of the ribosome. At this point, the putative “push” caused by the synthesis of peptide bonds must have ceased, and it is hard to imagine how a few residues on the luminal side could exert much of a “pull” on the peptide chain. The eventual passage through the membrane becomes all the more puzzling in view of the fact that the first 20 residues of melittin have largely hydrophobic side chains. Any theory of the mechanism by which growing polypeptide chains are threaded and propelled through the membrane of the endoplasmic reticulum must evidently take these facts into consideration. At least three enzymatic reactions must take place to convert prepromelittin to the end-product melittin: (1) cleavage of the prepeptide; (2) formation of the carboxy-terminal amide with concomitant removal of the extra glycine residue; and (3) cleavage of the pro-part. These reactions probably take place in this temporal sequence, since promelittin isolated from venom glands, like end-product melittin, terminates with glutaminamide.’ At present, very little is known about the second reaction. A carboxy-terminal amide is found in many peptide hormones and other physiologically active peptides, and the nature of the amidated amino acid varies widely. As discussed previously,’ it is our conjecture that this modification of the carboxyl end generally proceeds via precursors with an additional glycine residue. Originally, we speculated that the formation of a terminal amide might proceed via a transamidase-type of reaction whereby glycine is replaced by an NH,-group. All attempts to demonstrate such a reaction

Queen bee venom contains much less phospholipase than worker bee venom

Abstract Phospholipase A 2 in queen bee venom was measured by direct enzymatic analysis, by the wheal/flare reaction of an allergic individual, and by translation of the venom gland m RNA in a wheat germ system. These analyses showed that on a weight for weight basis, venom from queen bees contains no more than one fortieth of the phospholipase activity present in the venom from mature worker bees. The formation of phospholipase A 2 is thus subject to different control mechanisms in these castes of indentical genotype.

Substrate specificity of a peptidyl-aminoacyl-L/D-isomerase from frog skin.

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids.