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. 1998 Aug 1;333(Pt 3):539–542. doi: 10.1042/bj3330539

Evaluation of the stoichiometry and energetics of carbohydrate binding to Ricinus communis agglutinin: a calorimetric study.

S Sharma 1, S Bharadwaj 1, A Surolia 1, S K Podder 1
PMCID: PMC1219614  PMID: 9677310

Abstract

High-sensitivity isothermal titration calorimetry has been used to investigate the thermodynamics of binding of Ricinus communis agglutinin to galactose, lactose and their derivatives in the temperature range 280.5-298 K. The present study unequivocally establishes the carbohydrate-binding stoichiometry of the tetrameric agglutinin from castor bean as two, i.e. the (As-sB)2-type tetramer of the agglutinin has two equivalent sites that are non-interacting and independent. The site binding constants range from 2.2x10(3) M-1 at 282 K for galactose to 4.84x10(4) M-1 at 281 K for N-acetyl-lactosamine. The binding enthalpies range from -21.9 kJ. mol-1 at 293 K for 4-methylumbelliferyl-beta-galactoside to -50.2 kJ. mol-1 at 292.9 K for thiodigalactoside. The observation of limited entropy-enthalpy compensation for binding of the sugars to the lectin indicates that reorganization of water molecules plays an important role in binding. As the slope of the compensation plot is greater than unity, the reactions are largely enthalpically driven. These studies show that the stronger binding of N-acetyl-lactosamine than lactose is due to a favourable interaction between the acetamido group of the reducing-end N-acetylglucosamine of the former and the corresponding loci in the agglutinin molecule. Preferential binding of methyl-beta-galactoside over methyl-alpha-galactoside also indicates the apolar nature of the interaction with the methyl group of the former sugar.

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Selected References

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  1. Endo Y., Tsurugi K. RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem. 1987 Jun 15;262(17):8128–8130. [PubMed] [Google Scholar]
  2. Fisher H. F., Singh N. Calorimetric methods for interpreting protein-ligand interactions. Methods Enzymol. 1995;259:194–221. doi: 10.1016/0076-6879(95)59045-5. [DOI] [PubMed] [Google Scholar]
  3. Gupta D., Cho M., Cummings R. D., Brewer C. F. Thermodynamics of carbohydrate binding to galectin-1 from Chinese hamster ovary cells and two mutants. A comparison with four galactose-specific plant lectins. Biochemistry. 1996 Dec 3;35(48):15236–15243. doi: 10.1021/bi961458+. [DOI] [PubMed] [Google Scholar]
  4. Hatakeyama T., Ohba H., Yamasaki N., Funatsu G. Binding of saccharides to ricin E isolated from small castor beans. J Biochem. 1989 Mar;105(3):444–448. doi: 10.1093/oxfordjournals.jbchem.a122684. [DOI] [PubMed] [Google Scholar]
  5. Hegde R., Podder S. K. Studies on the variants of the protein toxins ricin and abrin. Eur J Biochem. 1992 Feb 15;204(1):155–164. doi: 10.1111/j.1432-1033.1992.tb16618.x. [DOI] [PubMed] [Google Scholar]
  6. Houston L. L., Dooley T. P. Binding of two molecules of 4-methylumbelliferyl galactose or 4-methylumbelliferyl N-acetylgalactosamine to the B chains of ricin and Ricinus communis agglutinin and to purified ricin B chain. J Biol Chem. 1982 Apr 25;257(8):4147–4151. [PubMed] [Google Scholar]
  7. Khan M. I., Mathew M. K., Balaram P., Surolia A. Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin. Biochem J. 1980 Nov 1;191(2):395–400. doi: 10.1042/bj1910395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. O'Hare M., Roberts L. M., Lord J. M. Biological activity of recombinant Ricinus communis agglutinin A chain produced in Escherichia coli. FEBS Lett. 1992 Mar 16;299(3):209–212. doi: 10.1016/0014-5793(92)80116-x. [DOI] [PubMed] [Google Scholar]
  9. Olsnes S., Fernandez-Puentes C., Carrasco L., Vazquez D. Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains. Eur J Biochem. 1975 Dec 1;60(1):281–288. doi: 10.1111/j.1432-1033.1975.tb21001.x. [DOI] [PubMed] [Google Scholar]
  10. Olsnes S., Saltvedt E., Pihl A. Isolation and comparison of galactose-binding lectins from Abrus precatorius and Ricinus communis. J Biol Chem. 1974 Feb 10;249(3):803–810. [PubMed] [Google Scholar]
  11. Podder S. K., Surolia A., Bachhawat B. K. On the specificity of carbohydrate-lectin recognition. The interaction of a lectin from Ricinus communis beans with simple saccharides and concanavalin A. Eur J Biochem. 1974 May 2;44(1):151–160. doi: 10.1111/j.1432-1033.1974.tb03468.x. [DOI] [PubMed] [Google Scholar]
  12. Roberts L. M., Lamb F. I., Pappin D. J., Lord J. M. The primary sequence of Ricinus communis agglutinin. Comparison with ricin. J Biol Chem. 1985 Dec 15;260(29):15682–15686. [PubMed] [Google Scholar]
  13. Rutenber E., Robertus J. D. Structure of ricin B-chain at 2.5 A resolution. Proteins. 1991;10(3):260–269. doi: 10.1002/prot.340100310. [DOI] [PubMed] [Google Scholar]
  14. Sphyris N., Lord J. M., Wales R., Roberts L. M. Mutational analysis of the Ricinus lectin B-chains. Galactose-binding ability of the 2 gamma subdomain of Ricinus communis agglutinin B-chain. J Biol Chem. 1995 Sep 1;270(35):20292–20297. doi: 10.1074/jbc.270.35.20292. [DOI] [PubMed] [Google Scholar]
  15. Van Wauwe J. P., Loontiens F. G., De Bruyne C. K. The interaction of Ricinus communis hemagglutinin with polysaccharides and low molecular weight carbohydrates. Biochim Biophys Acta. 1973 Jun 20;313(1):99–105. doi: 10.1016/0304-4165(73)90191-8. [DOI] [PubMed] [Google Scholar]
  16. Williams B. A., Chervenak M. C., Toone E. J. Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation. J Biol Chem. 1992 Nov 15;267(32):22907–22911. [PubMed] [Google Scholar]
  17. Wiseman T., Williston S., Brandts J. F., Lin L. N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem. 1989 May 15;179(1):131–137. doi: 10.1016/0003-2697(89)90213-3. [DOI] [PubMed] [Google Scholar]
  18. Zentz C., Frenoy J. P., Bourrillon R. Interaction entre l'hémagglutinine de ricin et ses ligands, galactose et lactose. Etude par microcalorimétrie et dialyse à l'équilibre. Biochimie. 1979;61(1):1–6. doi: 10.1016/s0300-9084(79)80307-7. [DOI] [PubMed] [Google Scholar]
  19. Zentz C., Frénoy J. P., Bourrillon R. Binding of galactose and lactose to ricin. Equilibrium studies. Biochim Biophys Acta. 1978 Sep 26;536(1):18–26. doi: 10.1016/0005-2795(78)90047-8. [DOI] [PubMed] [Google Scholar]

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