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Keith B, Foster NA, Bonettemaker M, Srivastava LM: In Vitro Gibberellin A (4) Binding to Extracts of Cucumber Hypocotyls. Plant Physiol. 1981 Aug;68(2):344-348.
Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3) H]-gibberellin (4) (GA (4)) 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 [(3) H] GA (4) to macromolecular components. [(3) H] GA (4) 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 [(3) H] GA (4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3) H] GA (4) 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. |
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