Biological and biochemical characterization of new basic phospholipase A(2) BmTX-I isolated from Bothrops moojeni snake venom.
BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.
Calgarotto
Andrana K
AK
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil.
Damico
Daniela C S
DC
Ponce-Soto
L A
LA
Baldasso
Paulo A
PA
Da Silva
Saulo L
SL
Souza
Gustavo H M F
GH
Eberlin
Marcos N
MN
Marangoni
Sergio
S
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D005591:Chemical Fractionation; D002645:Chickens / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology / Q000494:pharmacology; D017839:Crotalus / Q000378:metabolism; D003439:Crotoxin / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D019148:Neuromuscular Blockade; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003435:Crotalid Venoms; D009498:Neurotoxins; D003439:Crotoxin; C114084:myotoxin II, Bothrops moojeni; D010741:Phospholipases A