Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus)

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Abstract
Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 micrograms/ml and showed lethality in mice (LD50 = 1.2 micrograms/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2+. However, most of trimucrotoxin migrated as slowly as a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of crotoxin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A2. Furthermore, the purified acidic subunit of crotoxin potentiated the neurotoxicity of trimucrotoxin. A comparison of the sequences of these crotalid neurotoxins revealed some common features of the possible neurotoxic sites, including residues 6, 11, 76-81 and 119-125.

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