Soybean membranes possess high‐affinity binding sites for fungal β‐glucans that elicit phytoalexin synthesis. The ability of 1,3‐1,6‐β‐glucans, released by acid hydrolysis from mycelial walls of Phytophthora megasperma f.sp. glycinea, to compete for the putative phytoalexin elicitor receptors increases with their average degree of polymerization (DP). The results suggest a function where the probability for glucan fragments of containing a structural determinant that is optimal for binding approaches 1 as the DP tends to infinity. Ligand displacement data obtained against a 125I‐labeled glucan elicitor (average DP= 18) provided a theoretical minimum IC50 (50% inhibitory concentration) for 1,3‐1,6‐β‐glucans of 3 nM. The IC50 value obtained for a synthetic hepta‐β‐glucoside having a known elicitor‐active structure was 8 nM, remarkably close to the predicted value. Displacement of the 125I‐glucan of large DP was uniform and complete showing that the heptaglucoside had access, with similar affinity, to all sites available to the radioligand. Further analysis using a 125I‐labeled aminophenethylamine derivative of the heptaglucoside suggested that the putative glucan‐elicitor receptors bind a basic structural determinant present in all elicitor‐active glucans from the soybean pathogen P. megasperma.

A putative receptor protein for a hepta-B-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified P-glucan-binding proteins with a photolabile '251-labeled 2-(4-azidophenyl)ethylamino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 1251-labeling of the protein band by underivatized hepta-fi-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligandbinding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak '251-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/ PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.The perception mechanisms that signal the presence of potential pathogens and lead to the activation of defense responses in plant cells are still poorly understood. A representative case is that of the production of pterocarpan phytoalexins by soybean cells when infected by the pathogenic fungus Phytuphthora megasperma f.sp. glycineu. The phytoalexin response in soybean has been the object of detailed study (Ebel, 1986;Ebel and Grisebach, 1988). The accumulated information on the minimal structural requirements for phytoalexin-elicitor activity of P . megasperma mycelial-wall glucans (Sharp et al., 1984a, b; A commonly proposed model for signal recognition in the phytoalexin response involves a receptor located on the surface of plant cells that specifically binds phytoalexin elicitor(s) (Ebel, 1986;Dixon and Lamb, 1990). The best charac- 3) and (1,6)-linked-P-glucoside with high elicitor activity from mycelial-wall hydrolysates provided initial insight into the minimum structural requirements for elicitor activity in soybean. Additional information was obtained with the chemical synthesis of the hepta-fi-glucoside and comparison of its biological activity with that of a number of oligoglucosides of related structure (Sharp et al., 1984b;Ossowski et al., 1984;. Evidence for the existence of high-affinity-binding sites for P. megasperma (1,3) and (1,6)-linked P-glucan fragments in soybean membranes was provided initially by Schmidt and Ebel (1987) Additional work by our group defined optimal conditions for the solubilization, using detergents, of the glucan-binding proteins and for a partial purification of ...

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Multiple isotope effects have been measured for the acid-catalyzed hydrolysis of methyl formate in 0.5 M HCl at 20 degrees C. The isotope effects in the present investigation include the carbonyl carbon (13k = 1.028 +/- 0.001), the carbonyl oxygen (18k = 0.9945 +/- 0.0009), the nucleophile oxygen (18k = 0.995 +/- 0.001), and the formyl hydrogen ((D)k = 0.81 +/- 0.02). Determination of the carbonyl carbon, carbonyl oxygen, and formyl hydrogen isotope effects was performed via isotopic analysis of residual substrate. However, determination of the oxygen nucleophile isotope effect required analysis of the oxygen atoms of the product (formic acid), which exchange with the solvent (water) under acid conditions. This necessitated measurement of the rate of exchange of these oxygen atoms under the conditions for hydrolysis (k(ex) = 0.0723 min(-1)) and correction of the raw isotope ratios measured during the nucleophile-O isotope effect experiment. These results, along with the previously reported isotope effect for the leaving oxygen (18k = 1.0009) and the ratio of the rate of hydrolysis to that of exchange of the carbonyl oxygen with water (k(h)/k(ex) = 11.3), give a detailed picture of the transition-state structure for the reaction.