A new phospholipase A(2) isoform isolated from Bothrops neuwiedii (Yarara chica) venom with novel kinetic and chromatographic properties

A new phospholipase A(2) isoform, called P-3, isolated from Bothrops neuwiedii (Yarara chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity, In contrast to previously reported B. neuwiedii phospholipase A? isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide, The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers, P-3 had an optimum activity towards dialuroylphosphatidylcholine monolayers of 27 nM/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m, P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single close of 5 mu g/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 mu g/ml for P-3 and of 15 mu g/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 mu g/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 mu g/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2. (C) 1997 Elsevier Science Ltd.