In Vitro Gibberellin A4 Binding to Extracts of Cucumber Hypocotyls 1

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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 Kd of 10−7 molar and a concentration of 0.8 × 10−12 moles gram−1 fresh weight or 0.4 × 10−12 moles milligram−1 soluble protein.

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