Journal of Protein Chemistry

, Volume 21, Issue 3, pp 161–168 | Cite as

Structural and Functional Characterization of Basic PLA2 Isolated from Crotalus durissus terrificus Venom

  • D. G. Oliveira
  • M. H. Toyama
  • J. C. Novello
  • L. O. S. Beriam
  • S. Marangoni
Article

Abstract

The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA2 also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2 showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2 isoform, determined by automatic sequencing, was HLLQFNKMLKFETRKNAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2 isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2 cited in the literature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2 as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity.

Crotoxina bacteria enzyme kinetics sequencing PLA2 

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Copyright information

© Plenum Publishing Corporation 2002

Authors and Affiliations

  • D. G. Oliveira
    • 1
  • M. H. Toyama
    • 1
    • 2
  • J. C. Novello
    • 1
  • L. O. S. Beriam
    • 3
  • S. Marangoni
    • 1
  1. 1.Departamento de Bioquimica, Instituto de BiologiaUniversidade Estadual de Campinas (UNICAMP)Campinas, S. P.Brasil
  2. 2.Departamento de Fisiologia e Brofísica, Instituto de BiologiaUniversidade Estadual de Campinas (UNICAMP)Campinas, S. P.Brasil
  3. 3.Laboratório de Bacteriologia VegetalCentro Experimental do Instituto Biológico de CampinasCampinas, São Paulo, S. P.Brasil

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