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Abstract
The binding and phospholipase A2 activity of an 11,000-dalton β-bungarotoxin, isolated from Bungarus multicinctus venom, have been characterized using rat brain subcellular fractions as substrates. 1z51-labeled p-bungarotoxin binds rapidly (k = 0.14 min-l and 0.1 1 min-l), saturably (V max = 130.1 -+- 5.0 fmoles/mg and 128.2 ±7.1 fmoles/mg), and with high affinity (apparent KCI = 0.8 ± 0.1 nM and 0.7 ± 0.1 nM) to rat brain mitochondria and synaptosomal membranes, respectively, but not to myelin. The binding to synaptosomal membranes is inhibited by divalent cations and by pretreatment with trypsin. The binding results suggest that the toxin binds to specific protein receptor sites on presynaptic membranes. The 1 1,000-dalton toxin rapidly hydrolyzes synaptosomal membrane phospholipids to lysophosphatides and manifests relative substrate specificity in the order phosphatidyl ethanolamine > phosphatidyl choline > phosphatidyl serine. These results indicate that the 1 1,000-dalton p-bungarotoxin is a phospholipase A2 and can use presynaptic membrane phospholipids as substrates. The binding, phospholipase activity and other biological properties of the 1 1,000-dalton toxin are contrasted with those of the p-bungarotoxin found in highest concentration in the venom (the 22,000-dalton p-bungarotoxin), and the two toxins are shown to have qualitatively similar properties. Finally the results are shown to support the hypothesis that p-bungarotoxins act in a two-step fashion to inhibit transmitter release: first, by binding to a protein receptor site on the presynaptic membrane associated with Ca2+ entry, and second, by perturbing through enzymatic hydrolyses the phospholipid matrix of the membrane and thereby causing an increase in passive Ca2+ permeability.