Study of the interaction between different phosphodiesterases and yessotoxin using a resonant mirror biosensor.
Yessotoxins (YTXs) are disulfated polyether toxins that were first isolated from scallops in Japan. It has been proposed that these toxins activate cellular phosphodiesterases (PDEs). The interaction between YTX and PDEs was confirmed by resonant biosensor and fluorescence polarization studies. The aim of this work is to study the specificity of different PDEs for YTX binding. Association measurements are done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers from the sensor surface where the association PDEs-YTX takes place. We use aminosilane cuvettes, where exonuclease Phosphodiesterase I from Crotalus atrox (PDE I), exonuclease Phosphodiesterase II from bovine spleen (PDE II), or phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) are immobilized. Over immobilized exonuclease PDE I and exonuclease PDE II are added different amounts of YTX, and typical association curve profiles are observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration, the association rate constant (k(ass)) and the dissociation rate constant (k(diss)) are obtained. From these values, the kinetic equilibrium dissociation constant (K(D)) of the YTX-PDE association can be calculated, indicating the affinity between them. The specificity of cyclic nucleotide PDE families is studied using different inhibitors that are added over immobilized cyclic nucleotide PDEs. In these conditions, changes in the association PDEs-YTX curves are detected. The results show YTX affinity by cyclic nucleotide PDE 1, PDE 3, PDE 4, and exonuclease PDE I.
Pazos
María-José
MJ
Departamentos de Farmacología y Fisiología, and Area de Apoyo a la Investigación Agrobiológica, Facultad de Veterinaria, Universidad de Santiago, Lugo, Spain.
Alfonso
Amparo
A
Vieytes
Mercedes R
MR
Yasumoto
Takeshi
T
Botana
Luis M
LM
D000818:Animals; D015374:Biosensing Techniques; D002417:Cattle; D004800:Enzymes, Immobilized / Q000378:metabolism; D004988:Ethers, Cyclic / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D008978:Mollusk Venoms; D009712:Nucleotides, Cyclic / Q000378:metabolism; D035964:Oxocins / Q000737:chemistry / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding
D004800:Enzymes, Immobilized; D004988:Ethers, Cyclic; D008978:Mollusk Venoms; D009712:Nucleotides, Cyclic; D035964:Oxocins; D010727:Phosphoric Diester Hydrolases; C066632:yessotoxin