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 n euwiedii (Yarara chica) venom, showed different chromatographic, enzym atic and cytotoxic properties compared to the previously purified isof orms P-1 and P-2 but it had a similar edema-inducing activity, In cont rast to previously reported B. neuwiedii phospholipase A? isoforms, P- 3 did not interact with the oligosaccharide matrix of gel filtration c olumns (Superose, Superdex). Its molecular weight was 15,000 and its N -terminal 14 amino acid sequence was u-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met -Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an uniqu e histidine, presumably located at the active site, because a full inh ibition of enzymatic activity was observed after treatment with p-brom ophenacyl 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 2 7 nM/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an o ptimum of 13 mN/m with a cut-off of 22 mN/m, P-3 retained its edema-in ducing 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 b y 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 i soforms 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 inhi bited 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 t o 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.