Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom.
Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 mu-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I)-->Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35-45 degrees C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P<0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA2 was similar to another in the basic PLA2 of the crotoxin complex from C. durissus cascavella venom.
Damico
Daniela C S
DC
Department of Biochemistry, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil. dcdamico@unicamp.br
Lilla
Sérgio
S
de Nucci
Gilberto
G
Ponce-Soto
Luis A
LA
Winck
Flávia V
FV
Novello
José Camillo
JC
Marangoni
Sérgio
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D003439:Crotoxin / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D019216:Metals, Heavy / Q000378:metabolism; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D017422:Sequence Analysis, DNA; D017819:Viperidae
D003435:Crotalid Venoms; C072644:Lachesis venom; D019216:Metals, Heavy; D003439:Crotoxin; D010741:Phospholipases A; D054467:Phospholipases A2