Purification and primary structure of a myotoxic Lysine-49 phospholipase A₂ with low lipolytic activity from Trimeresurus gramineus venom

Four acidic phospholipase A₂ (PLA₂) isozymes named PLA₂-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced [Oda et al. (1991) Toxicon29, 157; Fukagawa et al. (1992) Toxicon30, 1131; Fukagawa et al. (1993) Toxicon 31, 957]. They contain aspartate-49 which is known to bind Ca²⁺, essential for catalysis. In the present study, a basic PLA₂ named PLA₂-V containing lysine-49 was newly isolated from the same snake venom. Its isoelectric point was 9.4 and considerably higher than those (c. 4.5) of PLA₂-I-IV. PLA₂-V was 1.1% as active as PLA₂-I toward egg-yolk emulsion but exhibited strong myotoxicity. The amino acid sequence of PLA₂-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages. PLA₂-V consists of 122 amino acid residues and is highly homologous (72-78%) to Lys-49 PLA₂s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA₂-I. The presence of Asn-28, which is characteristic of Lys-49 PLA₂s, was confirmed.