Sequence determination and characterization of a phospholipase A₂ isozyme from Trimeresurus gramineus (green habu snake) venom

In addition to phospholipase A₂-I (PLA₂-I) reported previously (Oda et al., 1991, Toxicon 29, 157), a new PLA₂ named PLA₂-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA₂ activity were conserved. Its sequence similarity to PLA₂-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA₂-I, PLA₂-II contains Tyr-28 as seen in most of other PLA₂s. There was no significant difference between the dissociation constants of PLA₂-I and PLA₂-II for Ca²⁺. Secondary structure compositions of PLA₂-II were similar to those of PLA₂-I and Crotalus atrox PLA₂. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA₂-II was over ten times more potent than PLA₂-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA₂-I. The difference in contractile activities of PLA₂-I and PLA₂-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA₂-I and Asn for PLA₂-II).