MOLECULAR-CLONING AND CHARACTERIZATION OF A NEUROTOXIC PHOSPHOLIPASE-A(2) FROM THE VENOM OF TAIWAN HABU (TRIMERESURUS-MUCROSQUAMATUS)

Using gel-filtration chromatography and reverse-phase (RP) HPLC we hav e purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its com plete primary structure was solved by an automated N-terminal sequenci ng and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 mu g/ml and showed lethality i n mice (LD(50) = 1.2 mu g/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filt ration experiment, and dissociates into monomers during SDS/PAGE in th e absence of Ca2+. However, most of trimucrotoxin migrated as slowly a s a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr 2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of croto xin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A(2). Furthermore, the pu rified acidic subunit of crotoxin potentiated the neurotoxicity of tri mucrotoxin. A comparison of the sequences of these crotalid neurotoxin s revealed some common features of the possible neurotoxic sites, incl uding residues 6, 11, 76-81 and 119-125.