Detection of abscisic-acid-binding proteins in the microsomal protein fraction of Arabidopsis thaliana with abscisic-acid-protein conjugates used as affinity probes.
A family of affinity probes has been generated to detect and purify abscisic-acid (ABA)-binding proteins, by coupling ABA onto carrier proteins (ovalbumin or BSA) through the C1 carboxyl group or the C4' carbonyl group of ABA. ELISA detection showed that these ABA-protein conjugates bound efficiently to the solubilized microsomal protein fraction of Arabidopsis thaliana, but not to the soluble protein fraction. Heat or proteolytic treatments inhibited the binding of the conjugates, indicating the protein nature of these binding sites. After membrane purification of the microsomes, the binding sites were found to be preferentially located in the plasma membrane fraction. The binding of the conjugates was independent of the nature of the carrier protein or the ABA-carrier protein linker, but was competitively inhibited with an anti-ABA mAb. Furthermore, the competitive inhibition of the binding of the conjugates with ABA, but not with the inactive ABA methyl ester analog, demonstrated the specificity of the binding and the saturability of the binding sites. The binding of the conjugates was strictly correlated to the ABA/carrier protein molar coupling ratio, confirming that the affinity of the conjugates to the ABA-binding proteins was enhanced by the increase in the probability of binding events. The experimental approach permits a new insight into the nature of membrane-associated ABA-binding proteins.
Pedron
J
J
Laboratoire de Physiologie du Développement des Plantes, URA CNRS 2135, Université Pierre et Marie Curie (Paris VI), France.
Brault
M
M
Nake
C
C
Miginiac
E
E
D000040:Abscisic Acid / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D017360:Arabidopsis / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis* / Q000302:isolation & purification; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D008861:Microsomes / Q000378:metabolism*; D010047:Ovalbumin; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D012710:Serum Albumin, Bovine
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D010940:Plant Proteins; D012710:Serum Albumin, Bovine; D000040:Abscisic Acid; D010047:Ovalbumin