Search

A 60kDa spider toxin binding protein from bovine brain was solubilized with digitonin and purified up to 5800-folds over starting crude homogenate. The purification procedure entailed DEAE-cellulose, concanavalin-A affinity, 1-naphthylacetyl spermine affinity and high performance liquid chromatography. The purified protein owned a very high affinity for ligand 125I-JSTX-3 binding Kd 15.6nM and Bmax 6.5nM. The amino acid composition of the protein was determined. The N-terminal amino acid sequence analysis yielded a unique sequence : NH2-X-Pro-X-Val-Tyr-Phe-Lys-Glu-Gln-Phe-Leu-Asp-Gly-Asp-X

Summary In our previous study on chemotactic peptide isolated from Ropalidian wasp, it was found to induce not only chemotaxis but also other cellular responses, such as superoxide generation and lysosomal enzyme release, but [Lys7] analog was found to induce only chemotaxis but not others. Here, studies on intracellular Ca2+ changes and receptor-binding revealed that the wasp chemotactic peptide and the authentic tripeptide stimulated two distinct receptors, but that the [Lys7] peptide could have access to only one of them.

1. At concentrations varying from 10(-8) to 10(-6) M synthetic poneratoxin (PoTX) is a strong, but very slowly acting agonist for smooth muscles and its blocks synaptic transmission in the insect CNS in a concentration-dependent manner and depolarizes giant interneurons. 2. However, in isolated dorsal unpaired median cells 10(-6) M PoTX causes only a reversible hyperpolarization of about 5 mV. 3. At concentrations from 10(-8) to 10(-6) M PoTX affects the electrical activity of isolated cockroach axons, as well as isolated frog and rat skeletal muscle fibres. 4. PoTX prolongs action potentials and induces slow automatic activity, due to a slow Na(+)-current activation at very negative values of potential and due to slow deactivation.

In a study on structure-activity relationship of spider toxin NSTX-3 obtained from venom of Nephila maculata, nine analogs or fragments of natural toxin were synthesized. Among them, des-Arg- NSTX-3 was found to be the minimum structure for blocking the excitatory transmission in the crustacean neuromuscular synapse. A positive charge of the arginine part was concluded to play the most important role for exhibition of the activity.

The release of free fatty acids from rat platelets, triggered by thrombin stimulation, was monitored by high-performance liquid chromatography (HPLC) after precolumn derivatization with monodansylcadaverine (MDC). A rapid filtration procedure was devised for the precise determination of free fatty acids released from aggregated platelets, instead of the conventional method using a stop solution or enzyme reactions. The fatty acids thus collected were derivatized with MDC in the presence of diethyl phosphorocyanidate (DEPC). The simultaneous separation of MDC derivatives of fatty acids was achieved on a reversed-phase TSKgel ODS-80 TM column within 60 min by linear gradient elution, using 0.2 M Tris-HCl buffer (pH 7.8)-methanol (50:50, v/v) and acetonitrile. The MDC derivatives were detected with excitation and emission wavelengths of 340 and 518 nm, respectively. The amounts of liberated fatty acids were in the range from 45.0 pmol for myristoleic acid (C 14:1 ) to 395.0 pmol for palmitic acid (C 16:0 ) per 1.9 · 10 7 platelets.

The planar structures of new eurocidin related compounds, eurocidins D and E, were elucidated from 1H-1H shift correlated 2D NMR spectra and other NMR data. All protons in the molecules were assigned. Eurocidins D and E have novel pentaenic structures of eurocidin family.

Solution conformations of Icaria chemotactic peptide isolated from the Ropalidian wasp venom and its [Lys7] analog have been analyzed by the use of two-dimensional proton nuclear resonance spectroscopy. It has been shown that Icaria chemotactic peptide takes α helical conformation in the C-terminal 8–13 segment with the N-terminal 1–7 segment disordered. By contrast, the [Lys7] analog takes α helical conformation over a wider range from residues 3 to 13. The present results indicate that the substitution of proline to lysine at position 7 results in a drastic change in solution conformation, providing the Icaria chemotactic peptide with new physiological functions.

Most of chemotactic peptides isolated from various kinds of wasp venom have lysine residue at 7th (or 8th) position, but only a chemotactic peptide from Icaria sp. has no basic amino acid residues in the sequence. The relation of chemotaxis with other biochemical activities such as superoxide generation and lysosomal enzyme release from guinea pig neutrophils was studied by the use of substitution analogs of Icaria chemotactic peptide at 7th position. Findings revealed two distinct ways of chemoattractant signal transduction in neutrophils.

1. The venom of the solitary scoliid wasp Colpa interrupta (F.) shows a kinin-activity, when tested on a cascade of mammalian smooth muscle preparations, and, in addition, a contraction of the rat colon. 2. The venom also irreversibly blocks the nicotinic synaptic transmission from the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach, Periplaneta americana . 3. The same activities have been found within one HPLC fraction. 4. However, rechromatography of this fraction resulted in four subfractions being active on smooth muscles. 5. One fraction caused contraction of the colon, three other fractions contained kinin-activity. 6. Only the most active kinin fraction blocked synaptic transmission in the insect CNS. 7. This fraction contained threonine 6 -bradykinin. 8. Synthetic Thr 6 -bradykinin causes irreversible presynaptic activation-induced block of transmission in the insect CNS.

The first total synthesis of Nephilatoxin-1 (NPTX-1), a Joro spider ( Nephila clavata ) toxin having a 4-hydroxyindole nucleus, has been achieved by using two key azide intermediates.

The structures of two regioisomes of tris(6-O-mesitylenesulfonyl)-α-cyclodextrin (6 A ,6 B ,6 D and 6 A ,6 B ,6 E ) were assigned by conversion of the isomers to tris(3,6-anhydro)-α-cyclodextrins and analyses of the two-dimensional 1 H NMR spectra (DQF-COSY, HOHAHA, and ROESY) of the anhydrocyclodextrins

The effective isolation of small peptides has been achieved by HPLC with alumina supports and hydrophilic eluents combined with a reversed phase HPLC. Of the four kinds of alumina supports tested, only Spherisorb A5Y showed the high column efficiency and a good reproducibility. Its elution mode appeared to be the same as the open column system, judging from the chromatograms of synthetic phyllolitorin and bradykinin. A mixture of the tryptic fragments of egg white lysozyme was separated by the combination of alumina and ODS columns, with a linear gradient elution of acetonitrile. The peaks which could not be isolated by one column were effectively separated by a second column; this was confirmed by determining their amino acid compositions. Spherisorb A5Y was found by the fluorescent X-ray analysis to be a complexed material of aluminum oxide and titanium oxide.

Effects of 10 synthetic compounds structurally related to spider toxins were studied on the glutamate receptors in crustacean neuromuscular synapses. The most potent synthetic compound was 1-naphthylacetyl spermine (approximately 1/10 of JSTX-3) which may be useful as a probe for studies on this type of glutamate receptor

The analysis of the flow-rate required to obtain the optimum height equivalent to a theoretical plate, HETP, has been used to estimate the molecular weight, MW, of peptides in size-exclusion chromatography. A straight line is obtained when log MW is plotted against the flow-rate which gives the optimum HETP. This relationship holds even for peptides which adsorb to column packings. The typical quantity of peptide required is 1 nmol and analysis time was less than 2 h. The method is applied to peptides of 200–10,000, and the precision is ±20%. The method may be applicable to the analysis of MW for all classes of compounds.

Synthetic Joro toxin (JSTX-3) was iodinated by the lactoperoxidase method and the reaction products were purified by high performance liquid chromatoqraphy. Proton-nuclear magnetic resonance spectroscopy revealed that iodination occurred mainly at position 3 of the benzene ring. The radio-iodinated JSTX-3 appeared autoradiographically to be accumulated in the synapse area of the lobster nerve-muscle preparation.

The effect of synthetic spider toxin analogs containing aza-crown in combination with zinc and copper was studied on the lobster neuromuscular synapse. The suppressive action of N4-type spider toxin analog (N4) on excitatory postsynaptic potentials (EPSPs) was markedly enhanced in the presence of 10(-5)-10(-4) M Zn2+. Cu2+ (10(-4) M) also had a potentiating effect on the suppression of EPSPs by N4 but to a lesser degree than Zn2+. While N6-type spider toxin analog (N6) suppressed EPSPs more effectively than N4, additional suppression was not pronounced in the presence of Zn2+ or Cu2+. The results suggest that chelatable divalent metal ions play an important role in the blocking mechanism of glutamate receptor by the spider toxin.

The chemical structure of angiotensin generated by incubating kidney extract with homologous plasma from the turtle, Pseudemys scripta, has been analyzed. The turtle angiotensin was proposed to be [Asp1, Val5, His9] ANG I by its amino acid composition and by its fluorescent peptide mapping. It was the same structure as angiotensin I in the ox and the sheep. The N-terminal amino acid of the turtle angiotensin was not blocked, unlike that of another reptilian angiotensin found in the snake, Elaphe climacophora.

Distribution of the binding sites of Joro spider toxin (JSTX), a specific inhibitor of the glutamate receptors in the crustacean neuromuscular synapse, was studied by using autoradiography. JSTX was synthesized and made radioactive by conjugation with iodine-125. 125I-JSTX irreversibly blocked the excitatory postsynaptic potentials of the lobster neuromuscular synapse in a similar manner as the natural spider toxin. Light microscopic autoradiography of 125I-JSTX treated muscle showed sporadic aggregates of reduced silver grains on the surface of muscles. Electron microscopy of adjoining ultrathin sections revealed that these spots corresponded to the fraction of sarcolemma apposed to axonal terminals with or without synaptic junctional profiles. This finding gives morphological support to the formulation that JSTX binds to the glutamate receptor-ion channel molecules.

Mastoparan, a peptide toxin from wasp venom, is a nonspecific secretagogue. We show here that mastoparan increases the GTPase activity and the rate of nucleotide binding of several purified GTP-binding regulatory proteins (G proteins) whose function is to couple cell-surface receptors to intracellular mediators. Mastoparan accelerated guanosine-5'-(3-O-thiotriphosphate binding and consequent G protein activation in part by promoting the dissociation of bound GDP, the mechanism by which receptors regulate G proteins. ADP-ribosylation by pertussis toxin, which uncouples receptors from G proteins, selectively inhibited mastoparan-stimulated activation. Like receptors, mastoparan was more potent if the G protein was reconstituted in phospholipid vesicles and was active at micromolar concentrations of Mg2+. The structure of mastoparan in a lipid bilayer is similar to that predicted for a cationic intracellular loop of G protein-coupled receptors. Mastoparan thus displays a novel mode of toxicity by acting directly on G proteins to mimic the role normally played by agonist-liganded receptors.

The fluorescent Kashland reagent, 2-carboxy-1-hydroxy-4-naphthylmethyldimethylsulfonium chloride (FKR), has three different functions : a) a phenolic hydroxy group ortho to carboxy group to chelate metal ions; b) a naphthyl moiety to produce fluorescence; and c) a Koshland reagent-type sulfonium chloride group to modify tryptophyl residues selectively in a peptide chain. Three procedures, methods I, II and III, using this reagent for the selective isolation of tryptophan-containing peptides from a protein digest were investigated. We selected lysozyme and carboxymethylated lysozyme as model proteins. In method I, lysozyme was labeled with FKR prior to the enzyme digestion, and the labeled protein was purified by gel permeation chromatography to remove the by-product formed from the reagent, then digested with protease. In method II, the protein (carboxymethylated lysozyme) was similarly labeled with the reagent, and the labeled protein was recovered as a precipitate by adding ethanol, leaving the excess reagent in the supernatant. The labeled protein was digested with protease. In method III, the protein was digested by protease before treatment with the reagent, and the by-product was removed by extraction with benzene. When the digests obtained by these procedures were applied to a Chelating Sepharose 6B column (Fe3+ or Al3+ form), FKR-labeled tryptophan-containing peptides were selectively adsorbed on the column owing to its chelating ability and could be eluted with 50 mM ethylenediaminetetraacetic acid. The final purification of these peptides was performed by reversed-phase high performance liquid chromatography on an octadecylsilane column in all cases.