In vivo Binding of Gibberellin A1 in Dwarf Pea Epicotyls

Abstract

Binding of [³H]gibberellin A₁ (GA₁) to extracts of dwarf pea epicotyls was investigated using sliced pea epicotyls (0.5-1.0 millimeter thick) that had been incubated in a solution containing [³H]GA₁ at 0 C for 3 days. Gel filtration of a 100,000g supernatant indicated binding to a high (HMW) and an intermediate molecular weight (IMW) fraction with estimated molecular weights of 6 × 10⁵ daltons and 4 to 7 × 10⁴ daltons, respectively. The bound ³H-activity was [³H]GA₁ and not a metabolite as deduced by thin layer chromatography. The bound label did not sediment during centrifugation at 100,000g for 2 hours; also, binding was not disrupted after treatment of a combined HMW and IMW fraction with DNase, RNase, or phospholipase A or C, but it was disrupted by protease or heat treatment. These facts suggest that binding of [³H]GA₁ was occurring to a soluble protein(s). [³H]GA₁ bound to a combined HMW and IMW fraction was not susceptible to changes in pH, nor could it be exchanged with a variety of GAs tested under in vitro conditions. Under in vivo equilibrium conditions, biologically active GAs, such as GA₁, GA₃, GA₄, GA₅, GA₇, and keto GA₁, could reduce the level of [³H]GA₁ binding, whereas inactive GAs, such as iodo GA₁ methyl ester, GA₈, GA₁₃, GA₂₆, and non-GAs, such as (±)abscisic acid, had no effect. By varying the concentration of [³H]GA₁ in the incubation medium, the specific binding of [³H]GA₁ appeared to be due to two classes of binding sites having estimated K_d of 6 × 10⁻⁸ molar and 1.4 × 10⁻⁶ molar. The concentrations of the two sites were estimated to be 0.45 picomole per gram and 4.04 picomoles per gram on a fresh weight and 0.1 picomole per milligram and 0.9 picomole per milligram on a soluble protein basis, respectively.