Cheong J J, Alba R, Côté F, Enkerli J, Hahn M G
University of Georgia, Complex Carbohydrate Research Center, Athens.
Plant Physiol. 1993 Dec;103(4):1173-82. doi: 10.1104/pp.103.4.1173.
Total membranes prepared from roots of soybean (Glycine max L.) seedlings have previously been shown to contain proteinaceous binding site(s) for a hepta-beta-glucoside elicitor of phytoalexin accumulation. The hepta-beta-glucoside elicitor-binding proteins have now been shown to co-migrate with a plasma membrane marker enzyme (vanadate-sensitive H(+)-ATPase) on linear sucrose density gradients. With the use of detergents, the elicitor-binding proteins have been solubilized in functional form from soybean root membranes. The nonionic detergents n-dodecylsucrose, n-dodecylmaltoside, and Triton X-114, at concentrations of 5 to 10 mg/mL, each solubilizes between 50 and 60% of the elicitor-binding activity in a single extraction of the membranes. A zwitterionic detergent, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZW 3-12), also solubilizes about 40% of the total binding activity at detergent concentrations between 1 and 2 mg/mL, but the total binding activity recovered is only approximately 50% of that recovered with the nonionic detergents. The elicitor-binding proteins solubilized with either n-dodecylsucrose or ZW 3-12 retain the high affinity for radiolabeled hepta-beta-glucoside elicitor (apparent dissociation constant [Kd] = 1.8 nM and 1.4 nM, respectively) that was observed with the membrane-localized binding proteins (apparent Kd = 1 nM). Competitive ligand-binding experiments with several structurally related synthetic oligoglucosides demonstrate that the solubilized binding proteins retain specificity for elicitor-active oligosaccharides, irrespective of the detergent used for solubilization. Moreover, the binding affinities of the oligoglucosides for the solubilized binding proteins correlate well with their abilities to induce phytoalexin accumulation in soybean cotyledon tissue. Gel-permeation chromatography of n-dodecylsucrose-solubilized elicitor-binding proteins demonstrate that the bulk of the elicitor-binding activity is associated with large detergent-protein micelles (relative molecular weight > 400,000). Our results suggest that n-dodecylsucrose is a suitable detergent for solubilizing elicitor-binding proteins from soybean root membranes with minimal losses of binding activity. More importantly, we demonstrate that solubilization does not significantly after the binding properties of the proteins for elicitor-active oligoglucosides.
先前已证明，从大豆（Glycine max L.）幼苗根部制备的总膜含有用于植物抗毒素积累的七-β-葡萄糖苷激发子的蛋白质结合位点。现已证明，七-β-葡萄糖苷激发子结合蛋白与质膜标记酶（钒酸盐敏感的H(+)-ATP酶）在线性蔗糖密度梯度上共同迁移。使用去污剂后，激发子结合蛋白已从大豆根膜中以功能形式溶解。非离子去污剂正十二烷基蔗糖、正十二烷基麦芽糖苷和Triton X-114，浓度为5至10 mg/mL时，在对膜进行单次提取时，每种去污剂都能溶解50%至60%的激发子结合活性。两性离子去污剂N-十二烷基-N,N-二甲基-3-铵基-1-丙烷磺酸盐（ZW 3-12）在去污剂浓度为1至2 mg/mL时，也能溶解约40%的总结合活性，但回收的总结合活性仅约为用非离子去污剂回收的50%。用正十二烷基蔗糖或ZW 3-12溶解的激发子结合蛋白对放射性标记的七-β-葡萄糖苷激发子保持高亲和力（表观解离常数[Kd]分别为1.8 nM和1.4 nM），这与膜定位的结合蛋白所观察到的情况（表观Kd = 1 nM）相同。用几种结构相关的合成寡糖苷进行的竞争性配体结合实验表明，无论用于溶解的去污剂如何，溶解的结合蛋白对激发子活性寡糖都保持特异性。此外，寡糖苷对溶解的结合蛋白的结合亲和力与其在大豆子叶组织中诱导植物抗毒素积累的能力密切相关。对正十二烷基蔗糖溶解的激发子结合蛋白进行凝胶渗透色谱分析表明，大部分激发子结合活性与大的去污剂-蛋白质胶束（相对分子质量>400,000）相关。我们的结果表明，正十二烷基蔗糖是一种合适的去污剂，用于从大豆根膜中溶解激发子结合蛋白，同时使结合活性损失最小。更重要的是，我们证明溶解不会显著改变蛋白质对激发子活性寡糖苷的结合特性。
