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Structural, enzymatic and biological properties of new PLA2 isoform from Crotalus durissus terrificus venom

2003; Elsevier BV; Volume: 41; Issue: 8 Linguagem: Inglês

10.1016/s0041-0101(03)00085-0

1879-3150

Marcos Hikari Toyama, Daniela Garcia de Oliveira, Luís Otávio Saggion Beriam, José C. Novello, Léa Rodrigues‐Simioni, Sérgio Marangoni,

Nicotinic Acetylcholine Receptors Study

We isolated a new PLA2 from the Crotalus durissus terrificus venom that designated F15, which showed allosteric behavior with a Vmax of 8.5 nmol/min/mg and a Km of 38.5 mM. The incubated heparin salt of this isolated F15 act a positive allosteric effector by increasing the Vmax to 10.2 nmol/min/mg, with decreasing the Vmax value to 20.5 mM. The crotapotin, on the other hand acts as a negative allosteric effector by increasing the Vmax values to 58.4 mM. F15 also showed high calcium dependence for its catalysis similar to that found for other PLA2 enzymes isolated from these snake venoms. The replacement of calcium by other divalent ions such Mg2+, Mn2+, Cd2+, Sn2+ and Cu2+ resulted in lower enzymatic activity. The optimum pH and temperature for the enzyme was 8.5 and 18 °C, respectively. F15 alone showed moderate neurotoxic activity in isolated mouse phrenic nerve diaphragm in comparison to other strong myotoxic PLA2 such as bothropstoxin-I (BThtx-I), but this activity was highly neurotoxic in a chick biventrer cervis preparation, whereas BthTx-I did not reveal this high neurotoxicity. This new protein showed a high bactericidal effect against both Gram-negative and Gram-positive bacterial strains. F15 contained 122 amino acid residues, with a primary structure of: HLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGQRRPKDATDRCCFVHDCCYGKLTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPKSRCRRPSETC. Its molecular mass and isoeletric point were 14.5 kDa and 8.85, both estimated by two dimensional electrophoresis. The amino acid sequence of the F15 revealed high sequence homology with F16 and F17. F15 and the other PLA2s revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. F15 also showed a high homology with the lysine-rich region of myotoxic PLA2.