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In Vitro Gibberellin A₄ Binding to Extracts of Cucumber Hypocotyls ¹

Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, V5A 1S6

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [³H]-gibberellin₄ (GA₄) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [³H]GA₄ to macromolecular components. [³H]GA₄ binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [³H]GA₄, and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [³H]GA₄ was due to a single class of binding sites having an estimated K_d of 10^–7 molar and a concentration of 0.8 x 10^–12 moles gram^–1 fresh weight or 0.4 x 10^–12 moles milligram^–1 soluble protein.