In Vitro Gibberellin A4 Binding to Extracts of Cucumber Hypocotyls

Abstract

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000 g to yield a supernatant or cytosol fraction. Binding of [3H]-GA4 to soluble macromolecular components present in the cytosol was demonstrated at 0.degree. 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 (NH4)2SO4 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 GA or other plant hormones such as IAA, abscisic acid or kinetin. TLC 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 M and a concentration of 0.8 .times. 10-12 mol g-1 fresh wt or 0.4 .times. 10-12 mol mg-1 soluble protein.