title: Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom.
abstract: A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40 degrees C. Full PLA2 activity required Ca2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+. The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu2+ also reduced the enzymatic activity. Bj V had PLA2 activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.
authors.affiliation: Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, (UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil.
authors.forename: Vera Luis
authors.initials: VL
authors.lastname: Bonfim
authors.forename: Luis Alberto
authors.initials: LA
authors.lastname: Ponce-Soto
authors.forename: Jose Camilo
authors.initials: JC
authors.lastname: Novello
authors.forename: Sergio
authors.initials: S
authors.lastname: Marangoni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000502:physiology*; D002460:Cell Line; D003435:Crotalid Venoms / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D032446:Myoblasts / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics / Q000502:physiology*; D054467:Phospholipases A2
chemical_list: D003435:Crotalid Venoms; D007527:Isoenzymes; D010741:Phospholipases A; D054467:Phospholipases A2