In vitro binding of gibberellin A4 to extracts of cucumber measured by using DEAE-cellulose filters

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RESUMO

A rapid method for assaying [3H]gibberellin A4 bound to a soluble protein from cucumber hypocotyls by using DEAE-cellulose filter discs is described. The binding is saturable, reversible with unlabeled gibberellin A4, and has a half-life of association under nonequilibrium conditions at 0-4°C of 6-7 min. By using this assay, the dissociation constant (Kd) was estimated to be 70 nM and the number of binding sites, 0.4 pmol mg-1 of soluble protein or 0.69 pmol g-1 (fresh weight) of hypocotyls. The speed and reliability of the assay make it invaluable for kinetic studies involving competitors. Thus, it has been possible to show that gibberellins that are biologically active in a cucumber bioassay compete for binding to the same protein and their calculated affinity constants bear a direct relationship to their known activities in the cucumber bioassay. Gibberellins that are inactive in this bioassay and other plant hormones, such as indoleacetic acid, abscisic acid, and kinetin, show a noncompetitive interaction.

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