Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (Maracambóia) venom.
The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degrees C. The C. d. cascavella PLA2 required Ca2+ for activity, but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors.
Beghini
D G
DG
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brazil.
Toyama
M H
MH
Hyslop
S
S
Sodek
L
L
Novello
J C
JC
Marangoni
S
S
D000494:Allosteric Regulation / Q000187:drug effects; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D003439:Crotoxin / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019216:Metals, Heavy / Q000494:pharmacology; D008970:Molecular Weight; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D021122:Protein Subunits; D013696:Temperature
D000596:Amino Acids; D003435:Crotalid Venoms; D019216:Metals, Heavy; D021122:Protein Subunits; D006493:Heparin; D003439:Crotoxin; D010741:Phospholipases A; D054467:Phospholipases A2