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. 1993 Dec;103(4):1173–1182. doi: 10.1104/pp.103.4.1173

Solubilization of functional plasma membrane-localized hepta-beta-glucoside elicitor-binding proteins from soybean.

J J Cheong 1, R Alba 1, F Côté 1, J Enkerli 1, M G Hahn 1
PMCID: PMC159103  PMID: 8290628

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 after the binding properties of the proteins for elicitor-active oligoglucosides.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Bruns R. F., Lawson-Wendling K., Pugsley T. A. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem. 1983 Jul 1;132(1):74–81. doi: 10.1016/0003-2697(83)90427-x. [DOI] [PubMed] [Google Scholar]
  3. Chanson A., Pilet P. E. Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes. Plant Physiol. 1987 Aug;84(4):1431–1436. doi: 10.1104/pp.84.4.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheong J. J., Birberg W., Fügedi P., Pilotti A., Garegg P. J., Hong N., Ogawa T., Hahn M. G. Structure-activity relationships of oligo-beta-glucoside elicitors of phytoalexin accumulation in soybean. Plant Cell. 1991 Feb;3(2):127–136. doi: 10.1105/tpc.3.2.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheong J. J., Hahn M. G. A specific, high-affinity binding site for the hepta-beta-glucoside elicitor exists in soybean membranes. Plant Cell. 1991 Feb;3(2):137–147. doi: 10.1105/tpc.3.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cosio E. G., Frey T., Ebel J. Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean. Eur J Biochem. 1992 Mar 15;204(3):1115–1123. doi: 10.1111/j.1432-1033.1992.tb16736.x. [DOI] [PubMed] [Google Scholar]
  7. Cosio E. G., Frey T., Ebel J. Solubilization of soybean membrane binding sites for fungal beta-glucans that elicit phytoalexin accumulation. FEBS Lett. 1990 May 21;264(2):235–238. doi: 10.1016/0014-5793(90)80256-i. [DOI] [PubMed] [Google Scholar]
  8. Cosio E. G., Pöpperl H., Schmidt W. E., Ebel J. High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts. Eur J Biochem. 1988 Aug 1;175(2):309–315. doi: 10.1111/j.1432-1033.1988.tb14198.x. [DOI] [PubMed] [Google Scholar]
  9. Gallagher S. R., Leonard R. T. Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots. Plant Physiol. 1982 Nov;70(5):1335–1340. doi: 10.1104/pp.70.5.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hahn M. G., Darvill A. G., Albersheim P. Host-Pathogen Interactions : XIX. THE ENDOGENOUS ELICITOR, A FRAGMENT OF A PLANT CELL WALL POLYSACCHARIDE THAT ELICITS PHYTOALEXIN ACCUMULATION IN SOYBEANS. Plant Physiol. 1981 Nov;68(5):1161–1169. doi: 10.1104/pp.68.5.1161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hill A. V. The Combinations of Haemoglobin with Oxygen and with Carbon Monoxide. I. Biochem J. 1913 Oct;7(5):471–480. doi: 10.1042/bj0070471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jin D. F., West C. A. Characteristics of galacturonic Acid oligomers as elicitors of casbene synthetase activity in castor bean seedlings. Plant Physiol. 1984 Apr;74(4):989–992. doi: 10.1104/pp.74.4.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lamb C. J., Lawton M. A., Dron M., Dixon R. A. Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell. 1989 Jan 27;56(2):215–224. doi: 10.1016/0092-8674(89)90894-5. [DOI] [PubMed] [Google Scholar]
  14. Leonard R. T., Vanderwoude W. J. Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll. Plant Physiol. 1976 Jan;57(1):105–114. doi: 10.1104/pp.57.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peters B. M., Cribbs D. H., Stelzig D. A. Agglutination of plant protoplasts by fungal cell wall glucans. Science. 1978 Jul 28;201(4353):364–365. doi: 10.1126/science.201.4353.364. [DOI] [PubMed] [Google Scholar]
  16. Schmidt W. E., Ebel J. Specific binding of a fungal glucan phytoalexin elicitor to membrane fractions from soybean Glycine max. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4117–4121. doi: 10.1073/pnas.84.12.4117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sharp J. K., Albersheim P., Ossowski P., Pilotti A., Garegg P., Lindberg B. Comparison of the structures and elicitor activities of a synthetic and a mycelial-wall-derived hexa(beta-D-glucopyranosyl)-D-glucitol. J Biol Chem. 1984 Sep 25;259(18):11341–11345. [PubMed] [Google Scholar]
  18. Sharp J. K., McNeil M., Albersheim P. The primary structures of one elicitor-active and seven elicitor-inactive hexa(beta-D-glucopyranosyl)-D-glucitols isolated from the mycelial walls of Phytophthora megasperma f. sp. glycinea. J Biol Chem. 1984 Sep 25;259(18):11321–11336. [PubMed] [Google Scholar]
  19. Sharp J. K., Valent B., Albersheim P. Purification and partial characterization of a beta-glucan fragment that elicits phytoalexin accumulation in soybean. J Biol Chem. 1984 Sep 25;259(18):11312–11320. [PubMed] [Google Scholar]
  20. Yoshikawa M., Keen N. T., Wang M. C. A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation. Plant Physiol. 1983 Oct;73(2):497–506. doi: 10.1104/pp.73.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]

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