Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions

The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50 pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of a least four protein components that are similar in their molecular weights (about 130,000) and isoelectric points (ranging from pH 5.2 to 5.5) and are immunologically indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.     

beta-Bungarotoxin antagonizes the effect of alpha-latrotoxin from black widow spider venom on the neuromuscular junction

Sustained contraction of the chick biventer cervicis nerve-muscle preparations evoked by alpha-latrotoxin was antagonized quickly by beta-bungarotoxin. This effect of beta-bungarotoxin was dependent on its phospholipase A2 activity. In contrast, pancreatic phospholipase A2 was ineffective even at a much higher dose. It is concluded that alpha-latrotoxin needs intact presynaptic membrane to exert its effect.     

Effect of black widow spider venom on the cockroach heart

The effect of black widow spider venom [crude gland extract (CGE), gland lumen venom (GLV), or from direct bite] on the cardiac activity of Periplaneta americana was assayed both in vivo and in vitro. It was shown that these different forms of venom in all cases blocked the cockroach heart-beat. Both CGE and GLV showed a selective activity on the heart function compared with their effect on the CNS. It is suggested that cardiac block is due to impairment of either the cardiac nerve ganglia function or the myocardial neuromuscular junctions, or of both of them. The mode of action of the toxic effects on the heart is discussed. Experiments with antiserum from CGE indicate that the antigenic fractions of CGE and GLV affecting the heart function, as well as those affecting the insect motor functions, are common to both toxic materials.     

Effects of black widow spider venom and Ca2+ on quantal secretion at the frog neuromuscular junction

A modification of the classical procedure of fluctuation analysis is used to measure the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of miniature endplate potentials (MEPPs) at frog cutaneous pectoris neuromuscular junctions treated with black widow spider venom (BWSV). MEPP parameters are determined from the power spectrum of the fluctuating potential and the second (variance), third (skew), and fourth semi-invariants (cumulants) of high-pass-filtered records of the potential. The method gives valid results even when the mean potential undergoes slow changes unrelated to MEPPs and when the MEPP rate is not stationary; it detects changes in the distribution of MEPP amplitudes and corrects for the nonlinear summation of MEPPs. The effects of Ca2+ on BWSV-induced secretion are studied in detail. When Ca2+ is absent, the power spectrum of the fluctuations is shaped like the spectrum of w(t) and secretion is quasi-stationary; (r) rises smoothly to peak values of approximately 1,500/s and then quickly subsides to levels near 10/s. Many relatively small and some "giant" MEPPs occur at the ends of the experiments, and the distribution of MEPP amplitudes broadens. When the effects of this broadening are corrected for, we find that approximately 0.7 X 10(6) MEPPs occurred during the 30 min of intense secretion. Since BWSV depletes nerve terminals of their quanta of transmitter and their synaptic vesicles, this figure is an upper limit for the quantal store in a resting terminal. When Ca2+ is present, the noise spectrum deviates from the spectrum of w(t) and secretion is nonstationary; (r) rises to similar peak values but is sustained at levels near 400/s for up to an hour and at least 1.5 X 10(6) quanta are secreted within this period. Thus, the quantal store must have turned over at least twice under this condition. Data previously obtained at junctions treated with La3+ are corrected for nonlinear summation and for the distribution of MEPP amplitudes. The two corrections roughly compensate each other, and the corrected results confirm the previous conclusion that the number of quanta secreted from La3+-treated terminals during 1 h is not strongly dependent upon the extracellular concentration of Ca2+; approximately 2 X 10(6) quanta are released even when Ca2+ is absent.     

Cloning and structure of cDNA encoding alpha-latrotoxin from black widow spider venom

cDNA encoding the putative alpha-latrotoxin precursor was isolated from spider venom glands cDNA library and sequenced. The cDNA contained the 4203 base-pair open reading frame corresponding to the 156,855-Da protein composed of 1401 amino acids. Computer analysis of the deduced primary structure revealed the presence of various internal imperfect repeats mainly in its central and C-terminal regions.     

The ionic dependence of black widow spider venom action at the stretch receptor neuron and neuromuscular junction of crustaceans

The effects of black widow spider venom (BWSV) on the crayfish stretch receptor and the lobster neuromuscular junction were examined. In crayfish stretch receptor neurons, BWSV caused a slight hyperpolarization followed by a large depolarization. The venom-induced depolarization of the strech receptor was caused by an increase in membrane conductance to Na⁺ and Ca²⁺. Black widow spider venom also caused an increase in the frequency of miniature inhibitory postsynaptic potentials recorded in the strech receptor. The ability of BWSV to increase the frequency of miniature excitatory postsynaptic potentials (MEPSPs) at the lobster neuromuscular junction was dependent on the divalent cation composition of the bathing medium. Ringer solutions containing Ca²⁺ supported the greatest venom-induced increase in MEPSP frequency, Mg²⁺ and Mn²⁺ supported a moderate increase in MEPSP frequency, while Co²⁺ and Zn²⁺ blocked this venom effect entirely. Black widow spider venom did not block axonal conduction in lobster walking leg axons or in the axon of the crayfish stretch receptor. The results suggest that in crustaceans, BWSV interacts specifically with membrane of the soma-dendritic region of the stretch receptor and with nerve terminal membrane, causing an increase in Na⁺ and Ca²⁺ conductance.     

Structure of tryptic fragments of a neurotoxin from black widow spider venom

The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.     

Study of the amino acid sequence of latroinsectotoxin from black widow spider venom]

The N-terminal amino acid sequence of alpha-latroinsectotoxin from the venom of Latrodectus mactans tredecimguttatus was determined. Then the toxin was digested by trypsin and total or partial amino acid sequences of twenty-six tryptic peptides were established. This resulted in the structural information needed for the construction of probes followed by the cloning of the alpha-latroinsectotoxin structural gene.     

A crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus

A method of the isolation of a crustacea-specific neurotoxin from the venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and hydrophobic chromatography on Phenyl-Superose column has been developed. LD50 of the toxin has been elucidated.     

Molecular cloning and primary structure of cDNA fragment for alpha-latrocrustatoxin from black widow spider venom

A fragment of the structural gene of alpha-latrocrustotoxin, a new representative of latrotoxins from black widow spider venom, was cloned. The fragment (1191 bp) was obtained by means of PCR based on the data obtained by sequencing tryptic peptides of the toxin. The fragment codes for a 397-aa sequence. The encoded polypeptide is the C-terminal fragment of the toxin central domain that presumably contains a site responsible for the toxin species specificity. The structural similarity of this fragment to the corresponding fragments of other latrotoxins was studied.     

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.     

alpha Latrotoxin of the black widow spider venom opens a small, non-closing cation channel

alpha Latrotoxin, a presynaptically acting polypeptide neurotoxin, induces massive neurotransmitter release from both synapses of vertebrates and the neurosecretory cells of the line PC12, derived from a rat pheochromocytoma. Single PC12 cells, differentiated by treatment with nerve growth factor, were used to investigate by the patch-clamp technique i) the alterations of the resting cell conditions (membrane potential and resistance) and ii) the microscopic mechanism of the permeability changes that underly the response to alpha LTx. The toxin was found to open a channel, 15 pS in conductance, that is permeable to various cations (Na+, K+ and probably Ca2+) and has little tendency to close. This channel is different from the classical voltage- and receptor-operated channels present in PC12 cells, as well as from the large conductances induced by the toxin in artificial lipid membranes.     

Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus]

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.     

Cation-anion selectivity and conductivity of the channels formed by black widow spider whole venom in lipid bilayer

It is established that BWSV (black widow spider venom) is capable of forming ion channels in lipid bilayer (BPhM). This effect is minimal upon pH value close to 5.0 and is increased either upon oxidation or alkalization of the medium. The amplitudes of the channels are monotonously decreased upon the reduction of pH with simultaneous narrowing of their dispersion. The degree of the change of BPhM conductivity by concentration of BWSV is close to 2 at different pH. Apparently the absorption of the active component of venom on bilayer surface is accompanied by doubling of the degree of its aggregation. Almost ideal cation selectivity of BWSV channels (pH 7.5) is significantly decreased upon the increase in ionic strength. Ionic channel is thought to be a zone of interaction of lipid domains. Possible structure of such a channel is discussed.