Purification and properties of a presynaptically acting neurotoxin, mandaratoxin, from hornet (Vespa mandarinia)

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Abstract
A hornet (V. mandarinia) neurotoxin, mandaratoxin (MDTX), was purified by simple procedures with column chromatography made on Sephadex G-50 and CM-Sephadex by using an acetate buffer. The MW of homogeneous MDTX was calculated to be .apprx. 20,000 by gel filtration, sodium dodecylsulfate disc gel electrophoresis and amino acid analysis. MDTX was a single-chain polypeptide. MDTX did not migrate electrophoretically in a basic buffer at pH 8.3 but did so when the buffer was acidic, at pH 4.3. The isoelectric point of the toxin was determined at 9.1 by isoelectric focusing. A relatively high amount of Lys was found in the amino acid analysis. .**GRAPHIC**. was 15.1. Glucosamine and galactosamine were not detectable by amino acid analysis. MDTX had no hemolytic or enzymatic activity. The toxin was heat labile. In neuromuscular junctions of a lobster walking leg, the nanomole range of MDTX irreversibly blocked the excitatory postsynaptic potential without appreciable change in the resting conductance of the postsynaptic membrane. Intracellular recording from the presynaptic nerve fiber showed that MDTX blocked the action potential mainly by reducing the Na current.