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

Abstract

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 at least four protein components that are similar in their molecular weights (about 130,000) and isoclectric points (ranging from pH 5.2 to 5.5) and are immunologicaUy 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.The physiological effects of the extract of the venom glands of the black widow spider, Latrodectus mactans, particularly the variety tredecimguttatus, have been studied extensively and the primary effects seem to be exerted on the nervous system (2-5, 7, 8, 12, 13, 15-18, 20-23, 30, 31, 33, 36-40, 43). Some of the active agents are proteins (18), and they seem to act in at least two basic ways: (a) to depolarize the cell bodies of some neurons and (b) to cause the release of neurotransmitters from a variety of nerve endings. For example, the extract depolarizes the cell body of the crayfish stretch receptor (S. Obara and A. Mauro, unpublished data) and induces a discharge of impulses in the axon (26). This depolarizing action of the extract may be responsible for its ability to induce a discharge of action potentials in the abdominal ganglion of the cockroach (5, 17).In addition to depolarizing excitable cells, the extract causes a release of transmitter from cholinergic nerve endings in brain (21, 24), sympathetic ganglia (38, 39), and Torpedo electric tissue (25), and from adrenergic nerve endings in the iris and other tissues (20,22,24). At neuromuscular junctions, the extract increases the frequency of occurrence of miniature end plate potentials, and blocks neuromuscular transmission. The neuromuscular effects have been observed at the cholinergic junctions of frogs and mammals (31, 37), at adrenergic nerve endings (vas deferens) in mammals (27), at both the inhibitory (3,-aminobutyrate) 462

Cholinergic Nerve Terminals in the Central Nervous System of Insects

“…It has been found that the venom of the black widow spider contains not only the well-characterized a-latrotoxin, (a-LTx), but also a fraction that appears to be toxic only t o arthropods (96). The primary effect of a-LTx is the stimulation of massive transmitter release with irreversible depletion of transmitter reserves (97).…”

Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane.

“…A class of neurotoxins is known to act presynaptically (5,15,22,27). Among these, a-latrotoxin (a-LTx),' from the venom of the black widow spider, was found to act on many, and possibly all, types of vertebrate synapses, where it induces massive stimulation of quantal release of neurotransmitter accompanied by parallel depletion of synaptic vesicles (9,16,22,24,29,41,45). In contrast, a-LTx is completely inactive on axons and on nonneural cells, irrespective oftheir specific properties (secretion, excitability, etc .)…”

High Affinity Binding of α-Latrotoxin to Recombinant Neurexin Iα

“…␣-Latrotoxin stimulates neurotransmitter release from vertebrate nerve terminals by triggering massive exocytosis of small synaptic vesicles (1,2). ␣-Latrotoxin-stimulated neurotransmitter release is accompanied by presynaptic membrane depolarization and the influx of Ca 2ϩ through ion channels induced by the toxin (3,4).…”