Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom.
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.
Bonfim
Vera Luis
VL
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, (UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil.
Ponce-Soto
Luis Alberto
LA
Novello
Jose Camilo
JC
Marangoni
Sergio
S
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
D003435:Crotalid Venoms; D007527:Isoenzymes; D010741:Phospholipases A; D054467:Phospholipases A2