Isolation And Enzymatic Characterization Of A Basic Phospholipase A2 From Bothrops Jararacussu Snake Venom

A novel basic phospholipase A2 (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj IV had high PLA2 activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA2 activity required Ca2+ but was inhibited by Cu2+ and Zn2+, and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA2 was similar to that in the basic PLA2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA2 could partly explain the low PLA2 activity of Bothrops venoms.203239245Arni, R.K., Ward, R.J., (1996) Toxicon, 34, pp. 827-841Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Bülbring, E., (1946) Br. J. Pharm., 1, pp. 39-61Chang, L.S., Kuo, K.W., Lin, S.R., Chang, C.C., (1994) J. Protein Chem., 13, pp. 641-648Cho, W., Kézdy, F.J., (1991) Meth. Enzymol., 197, pp. 75-79Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., (1993) J. Protein Chem., 12, pp. 57-64Daniele, J.J., Bianco, I.D., Delgado, C., Carrillo, D.B., Fidelio, G.D., (1997) Toxicon, 35, pp. 1205-1215Dennis, E.A., (1994) J. Biol. Chem., 269, pp. 13057-13060Fletcher, J.E., Jiang, M.S., (1996) Toxicon, 33, pp. 1565-1576Francis, B., Gutiérrez, J.M., Lomonte, B., Kaiser, J.I., (1991) Arch. Biochem. Biophys., 284, pp. 352-359Gutiérrez J., M., Lomonte, B., (1995) Toxicon, 33, pp. 1405-1424Habermann, E., Breithaupt, H., (1978) Toxicon, 16, pp. 19-30Heinrikson, R.L., Meredith, S.C., (1984) Anal. Biochem., 136, pp. 60-65Henrikson, R.L., Trueger, E.T., Kein, P.S., (1977) J. Biol. Chem., 252, pp. 4913-4921Holzer, M., Mackessy, S.P., (1996) Toxicon, 35, pp. 1149-1155Homsi-Brandeburgo, M.I., Queiroz, L.S., Santo-Neto, H., Rodrigues-Simioni, L., Giglio, J.R., (1988) Toxicon, 26, pp. 615-627Kini, R.M., Evans, H.J., (1989) Int. J. Peptide Protein Res., 34, pp. 277-286Landucci, E.C.T., De Castro, R.C., Toyama, M., Giglio, J.R., Marangoni, S., De Nucci, G., Antunes, E., (2000) Biochem. Pharmacol., 59, pp. 1289-1294Mancuso, L.C., Correa, M.M., Vieira, C.A., Cunha, O.A.B., Lachat, J.J., Selistre, H.S.S., Ownby, C.L., Giglio, J.R., (1995) Toxicon, 33, pp. 615-626Ogawa, T., Nakashima, K.I., Nobushima, I., Deshimaru, M., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Ohno, M., (1996) Toxicon, 34, pp. 1229-1236Pereira, M.E., Novello, J.C., Cintra, A.C.O., Giglio, J.R., Landucci, E.T., Oliveira, B., Marangoni, S., (1998) J. Protein Chem., 17, pp. 381-386Schägger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Selistre de Araújo, H.S., White, S.P., Ownby, C.L., (1996) Toxicon, 34, pp. 1237-1242Shimohigashi, Y., Tani, A., Matsumoto, H., Nakashima, K.I., Yamaguchi, Y., Oda, N., Takano, Y., Ohno, M., (1995) J. Biochem., 118, pp. 1037-1044Soares, A.M., Rodriques, V.M., Homsi-Brandeburgo, M.H., Toyama, M.H., Lombard, F.R., Arni, A.K., Giglio, J.R., (1998) Toxicon, 36, pp. 503-514Toyama, M.H., Mancuso, L.C., Giglio, J.R., Novello, J.C., Oliveira, B., Marangoni, S., (1995) Biochem. Mol. Biol. Int., 37, pp. 1047-1055Toyama, M.H., Costa, P.D., Novello, J.C., De Oliveira, B., Giglio, J.R., Da Cruz-Hofling, M.A., Marangoni, S., (1999) J. Protein Chem., 18, pp. 371-37