Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi.
A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
J W
JW
Govers-Riemslag
M J
MJ
Knapen
G
G
Tans
R F
RF
Zwaal
J
J
Rosing
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005165:Factor V / Q000378:metabolism; D015943:Factor Va; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D010743:Phospholipids / Q000378:metabolism; D011092:Polyethylene Glycols; D011480:Protease Inhibitors / Q000494:pharmacology; D011516:Prothrombin / Q000378:metabolism*
D003435:Crotalid Venoms; D010743:Phospholipids; D011480:Protease Inhibitors; D011092:Polyethylene Glycols; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D017830:Octoxynol; D002118:Calcium