Keith B, Srivastava L M
Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada, V5A 1S6.
Plant Physiol. 1980 Nov;66(5):962-7. doi: 10.1104/pp.66.5.962.
Binding of [(3)H]gibberellin A(1) (GA(1)) 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 [(3)H]GA(1) 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 x 10(5) daltons and 4 to 7 x 10(4) daltons, respectively. The bound (3)H-activity was [(3)H]GA(1) 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 [(3)H]GA(1) was occurring to a soluble protein(s). [(3)H]GA(1) 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 GA(1), GA(3), GA(4), GA(5), GA(7), and keto GA(1), could reduce the level of [(3)H]GA(1) binding, whereas inactive GAs, such as iodo GA(1) methyl ester, GA(8), GA(13), GA(26), and non-GAs, such as (+/-)abscisic acid, had no effect. By varying the concentration of [(3)H]GA(1) in the incubation medium, the specific binding of [(3)H]GA(1) appeared to be due to two classes of binding sites having estimated K(d) of 6 x 10(-8) molar and 1.4 x 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.
使用在0℃下于含有[³H]赤霉素A₁（GA₁）的溶液中孵育3天的豌豆上胚轴切片（0.5 - 1.0毫米厚），研究了[³H]GA₁与矮豌豆上胚轴提取物的结合情况。对100,000g上清液进行凝胶过滤表明，它与一个高分子量（HMW）组分和一个中分子量（IMW）组分结合，估计分子量分别为6×10⁵道尔顿和4至7×10⁴道尔顿。结合的³H活性是[³H]GA₁，而非如薄层色谱所推断的代谢物。结合的标记物在100,000g离心2小时期间不会沉淀；此外，用DNA酶、RNA酶、磷脂酶A或C处理HMW和IMW混合组分后，结合未被破坏，但蛋白酶处理或热处理会破坏结合。这些事实表明[³H]GA₁正在与一种可溶性蛋白质结合。与HMW和IMW混合组分结合的[³H]GA₁对pH变化不敏感，在体外条件下也不能与多种测试的赤霉素进行交换。在体内平衡条件下，生物活性赤霉素，如GA₁、GA₃、GA₄、GA₅、GA₇和酮基GA₁，可降低[³H]GA₁的结合水平，而无活性的赤霉素，如碘代GA₁甲酯、GA₈、GA₁₃、GA₂₆，以及非赤霉素，如（±）脱落酸，则没有影响。通过改变孵育培养基中[³H]GA₁的浓度，[³H]GA₁的特异性结合似乎归因于两类结合位点，估计解离常数（Kd）分别为6×10⁻⁸摩尔和1.4×10⁻⁶摩尔。以鲜重计，这两类位点的浓度估计分别为每克0.45皮摩尔和4.04皮摩尔，以可溶性蛋白质计，分别为每毫克0.1皮摩尔和0.9皮摩尔。
