Solubilization of Functional Plasma Membrane-Localized Hepta-[beta]-Glucoside Elicitor-Binding Proteins from Soybean

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Abstract
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 alter the binding properties of the proteins for elicitor-active oligoglucosides.