Isolation and physiological characterization of taicatoxin, a complex toxin with specific effects on calcium channels.

Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an alpha-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in beta-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 micrograms of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only alpha-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the alpha-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The alpha-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.