Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance.
Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS-PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (K(d)(app)) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (k(off)=1.2 x 10(-2)s(-1)) and the association rate constant (k(on)=3.5 x 10(6)M(-1)s(-1)).
Faure
Grazyna
G
Unité des Venins, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris Cedex 15 France. fgrazyna@pasteur.fr
Copic
Alenka
A
Le Porrier
Sabine
S
Gubensek
Franc
F
Bon
Cassian
C
Krizaj
Igor
I
D000818:Animals; D017839:Crotalus; D003439:Crotoxin / Q000378:metabolism*; D004557:Electric Organ / Q000737:chemistry*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013570:Synaptic Membranes / Q000378:metabolism; D014101:Torpedo
D011956:Receptors, Cell Surface; D003439:Crotoxin