Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Mar 1;338(Pt 2):273–279.

Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding.

J Krupakar 1, C P Swaminathan 1, P K Das 1, A Surolia 1, S K Podder 1
PMCID: PMC1220052  PMID: 10024502

Abstract

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.

Full Text

The Full Text of this article is available as a PDF (164.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beaumelle B., Alami M., Hopkins C. R. ATP-dependent translocation of ricin across the membrane of purified endosomes. J Biol Chem. 1993 Nov 5;268(31):23661–23669. [PubMed] [Google Scholar]
  2. Beaumelle B., Bensammar L., Bienvenüe A. Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes. J Biol Chem. 1992 Jun 5;267(16):11525–11531. [PubMed] [Google Scholar]
  3. Brandts J. F., Hu C. Q., Lin L. N., Mos M. T. A simple model for proteins with interacting domains. Applications to scanning calorimetry data. Biochemistry. 1989 Oct 17;28(21):8588–8596. doi: 10.1021/bi00447a048. [DOI] [PubMed] [Google Scholar]
  4. Bushueva T. L., Tonevitskii A. G., Kindt A., Franz H. Struktura toksichnogo belka-lektina iz omely-pri razlichnykh pH: issledovanie metodom sobstvennoi fluorestsentsii. Mol Biol (Mosk) 1988 May-Jun;22(3):628–634. [PubMed] [Google Scholar]
  5. Bushueva T. L., Tonevitsky A. G. The effect of pH on the conformation and stability of the structure of plant toxin-ricin. FEBS Lett. 1987 May 4;215(1):155–159. doi: 10.1016/0014-5793(87)80132-1. [DOI] [PubMed] [Google Scholar]
  6. Chen J. K., Hung C. H., Liaw Y. C., Lin J. Y. Identification of amino acid residues of abrin-a A chain is essential for catalysis and reassociation with abrin-a B chain by site-directed mutagenesis. Protein Eng. 1997 Jul;10(7):827–833. doi: 10.1093/protein/10.7.827. [DOI] [PubMed] [Google Scholar]
  7. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  8. Frénoy J. P. Effect of physical environment on the conformation of ricin. Influence of low pH. Biochem J. 1986 Nov 15;240(1):221–226. doi: 10.1042/bj2400221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukada H., Sturtevant J. M., Quiocho F. A. Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli. J Biol Chem. 1983 Nov 10;258(21):13193–13198. [PubMed] [Google Scholar]
  10. Ganesh C., Shah A. N., Swaminathan C. P., Surolia A., Varadarajan R. Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein. Biochemistry. 1997 Apr 22;36(16):5020–5028. doi: 10.1021/bi961967b. [DOI] [PubMed] [Google Scholar]
  11. Goins B., Freire E. Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1. Biochemistry. 1988 Mar 22;27(6):2046–2052. doi: 10.1021/bi00406a035. [DOI] [PubMed] [Google Scholar]
  12. Hazes B., Read R. J. Accumulating evidence suggests that several AB-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells. Biochemistry. 1997 Sep 16;36(37):11051–11054. doi: 10.1021/bi971383p. [DOI] [PubMed] [Google Scholar]
  13. Hegde R., Maiti T. K., Podder S. K. Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography. Anal Biochem. 1991 Apr;194(1):101–109. doi: 10.1016/0003-2697(91)90156-n. [DOI] [PubMed] [Google Scholar]
  14. Ishida B., Cawley D. B., Reue K., Wisnieski B. J. Lipid-protein interactions during ricin toxin insertion into membranes. Evidence for A and B chain penetration. J Biol Chem. 1983 May 10;258(9):5933–5937. [PubMed] [Google Scholar]
  15. Ladbury J. E., Kishore N., Hellinga H. W., Wynn R., Sturtevant J. M. Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry. Biochemistry. 1994 Mar 29;33(12):3688–3692. doi: 10.1021/bi00178a027. [DOI] [PubMed] [Google Scholar]
  16. London E. Diphtheria toxin: membrane interaction and membrane translocation. Biochim Biophys Acta. 1992 Mar 26;1113(1):25–51. doi: 10.1016/0304-4157(92)90033-7. [DOI] [PubMed] [Google Scholar]
  17. Manly S. P., Matthews K. S., Sturtevant J. M. Thermal denaturation of the core protein of lac repressor. Biochemistry. 1985 Jul 16;24(15):3842–3846. doi: 10.1021/bi00336a004. [DOI] [PubMed] [Google Scholar]
  18. Montesano L., Cawley D., Herschman H. R. Disuccinimidyl suberate cross-linked ricin does not inhibit cell-free protein synthesis. Biochem Biophys Res Commun. 1982 Nov 16;109(1):7–13. doi: 10.1016/0006-291x(82)91558-3. [DOI] [PubMed] [Google Scholar]
  19. Olsnes S. Toxic and nontoxic lectins from Abrus precatorius. Methods Enzymol. 1978;50:323–330. doi: 10.1016/0076-6879(78)50036-0. [DOI] [PubMed] [Google Scholar]
  20. Pabo C. O., Sauer R. T., Sturtevant J. M., Ptashne M. The lambda repressor contains two domains. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1608–1612. doi: 10.1073/pnas.76.4.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Privalov P. L., Khechinashvili N. N. A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol. 1974 Jul 5;86(3):665–684. doi: 10.1016/0022-2836(74)90188-0. [DOI] [PubMed] [Google Scholar]
  22. Privalov P. L. Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem. 1982;35:1–104. [PubMed] [Google Scholar]
  23. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  24. Ramalingam T. S., Das P. K., Podder S. K. Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer. Biochemistry. 1994 Oct 11;33(40):12247–12254. doi: 10.1021/bi00206a030. [DOI] [PubMed] [Google Scholar]
  25. Ramsay G., Montgomery D., Berger D., Freire E. Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition. Biochemistry. 1989 Jan 24;28(2):529–533. doi: 10.1021/bi00428a018. [DOI] [PubMed] [Google Scholar]
  26. Rapak A., Falnes P. O., Olsnes S. Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3783–3788. doi: 10.1073/pnas.94.8.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Refsnes K., Olsnes S., Pihl A. On the toxic proteins abrin and ricin. Studies of their binding to and entry into Ehrlich ascites cells. J Biol Chem. 1974 Jun 10;249(11):3557–3562. [PubMed] [Google Scholar]
  28. Robertson Andrew D., Murphy Kenneth P. Protein Structure and the Energetics of Protein Stability. Chem Rev. 1997 Aug 5;97(5):1251–1268. doi: 10.1021/cr960383c. [DOI] [PubMed] [Google Scholar]
  29. Ross P. D., Shrake A. Decrease in stability of human albumin with increase in protein concentration. J Biol Chem. 1988 Aug 15;263(23):11196–11202. [PubMed] [Google Scholar]
  30. Sandvig K., Olsnes S., Pihl A. Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells. J Biol Chem. 1976 Jul 10;251(13):3977–3984. [PubMed] [Google Scholar]
  31. Schwarz F. P., Puri K., Surolia A. Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus). J Biol Chem. 1991 Dec 25;266(36):24344–24350. [PubMed] [Google Scholar]
  32. Simpson J. C., Dascher C., Roberts L. M., Lord J. M., Balch W. E. Ricin cytotoxicity is sensitive to recycling between the endoplasmic reticulum and the Golgi complex. J Biol Chem. 1995 Aug 25;270(34):20078–20083. doi: 10.1074/jbc.270.34.20078. [DOI] [PubMed] [Google Scholar]
  33. Sugita T., Totsuka T., Saito M., Yamasaki K., Taga T., Hirano T., Kishimoto T. Functional murine interleukin 6 receptor with the intracisternal A particle gene product at its cytoplasmic domain. Its possible role in plasmacytomagenesis. J Exp Med. 1990 Jun 1;171(6):2001–2009. doi: 10.1084/jem.171.6.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Surolia A., Sharon N., Schwarz F. P. Thermodynamics of monosaccharide and disaccharide binding to Erythrina corallodendron lectin. J Biol Chem. 1996 Jul 26;271(30):17697–17703. doi: 10.1074/jbc.271.30.17697. [DOI] [PubMed] [Google Scholar]
  35. Sánchez-Ruiz J. M., López-Lacomba J. L., Cortijo M., Mateo P. L. Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin. Biochemistry. 1988 Mar 8;27(5):1648–1652. doi: 10.1021/bi00405a039. [DOI] [PubMed] [Google Scholar]
  36. Tahirov T. H., Lu T. H., Liaw Y. C., Chen Y. L., Lin J. Y. Crystal structure of abrin-a at 2.14 A. J Mol Biol. 1995 Jul 14;250(3):354–367. doi: 10.1006/jmbi.1995.0382. [DOI] [PubMed] [Google Scholar]
  37. Thorpe P. E., Blakey D. C., Brown A. N., Knowles P. P., Knyba R. E., Wallace P. M., Watson G. J., Wawrzynczak E. J. Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different cross-linking agents: antitumor effects, in vivo fate, and tumor cell mutants. J Natl Cancer Inst. 1987 Nov;79(5):1101–1112. [PubMed] [Google Scholar]
  38. 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]
  39. Yoshida T., Chen C. C., Zhang M. S., Wu H. C. Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp Cell Res. 1991 Feb;192(2):389–395. doi: 10.1016/0014-4827(91)90056-z. [DOI] [PubMed] [Google Scholar]
  40. Youle R. J., Neville D. M., Jr Kinetics of protein synthesis inactivation by ricin-anti-Thy 1.1 monoclonal antibody hybrids. Role of the ricin B subunit demonstrated by reconstitution. J Biol Chem. 1982 Feb 25;257(4):1598–1601. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES