Isolation and preliminary enzymatic characterization of a novel PLA2 from Crotalus durissus collilineatus venom.
A crotoxin homolog was purified from the Crotalus durissus collilineatus venom using molecular exclusion and reverse-phase HPLC. This crotoxin contained one PLA2 (Cdcolli III F6) and four crotapotin isoforms, whereas crotoxin from Crotalus durissus terrificus venom had three PLA2 iso forms and two crotapotin isoforms. SDS-PAGE showed that the C. d. collilineatus PLA2 and crotapotin had relative molecular mass of 15 and 9 kDa, respectively. Neither the PLA2 (Cdcolli III F6) nor the crotapotins (Cdcolli III F3 and F4) had any neurotoxicity in mouse phrenic nerve-diaphragm preparations when tested alone. However, when PLA2 and crotapotin were coincubated before testing, the neurotoxicity was restored to a level similar to test in the venom in native crotoxin. The two crotapotins (Cdcolli III F3 and F4) differed in their ability to inhibit PLA2 activity, perhaps because of variations in their affinities for this enzyme. Cdcolli III F6 showed allosteric enzymatic behavior, with maximal activity at pH 8.3 and 36 degrees C. Full PLA2 activity required the presence of a low Ca2+ concentration and was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. These results indicate that crotoxin from C. d. collineatus venom is very similar enzymatically to crotoxin from C. d. terrificus.
Ponce-Soto
L A
LA
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), SP, Brasil.
Toyama
M H
MH
Hyslop
S
S
Novello
J C
JC
Marangoni
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003435:Crotalid Venoms / Q000201:enzymology*; D017839:Crotalus / Q000378:metabolism; D003439:Crotoxin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009579:Nitrobenzoates / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D054467:Phospholipases A2; D010791:Phrenic Nerve / Q000187:drug effects; D017386:Sequence Homology, Amino Acid
C104459:4-nitro-3-(octanoyloxy)benzoic acid; D003435:Crotalid Venoms; D007527:Isoenzymes; D009579:Nitrobenzoates; D003439:Crotoxin; D010741:Phospholipases A; D054467:Phospholipases A2