Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Jul 15;317(Pt 2):509–514. doi: 10.1042/bj3170509

Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854-->Leu).

J M Salhany 1, R L Sloan 1, L M Schopfer 1
PMCID: PMC1217516  PMID: 8713079

Abstract

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56-->Glu within the cytoplasmic domain, and Pro-854-->Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4'-di-isothiocyanatodihydro-2, 2'-stilbenedisulphonate) to 'lysine A' (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'off' rate constant, but no difference was found between mutant and control when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854-->Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

Full Text

The Full Text of this article is available as a PDF (400.8 KB).

Selected References

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

  1. Bruce L. J., Anstee D. J., Spring F. A., Tanner M. J. Band 3 Memphis variant II. Altered stilbene disulfonate binding and the Diego (Dia) blood group antigen are associated with the human erythrocyte band 3 mutation Pro854-->Leu. J Biol Chem. 1994 Jun 10;269(23):16155–16158. [PubMed] [Google Scholar]
  2. Casey J. R., Lieberman D. M., Reithmeier R. A. Purification and characterization of band 3 protein. Methods Enzymol. 1989;173:494–512. doi: 10.1016/s0076-6879(89)73034-2. [DOI] [PubMed] [Google Scholar]
  3. Dix J. A., Verkman A. S., Solomon A. K. Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3. J Membr Biol. 1986;89(3):211–223. doi: 10.1007/BF01870665. [DOI] [PubMed] [Google Scholar]
  4. Eisinger J., Flores J., Salhany J. M. Association of cytosol hemoglobin with the membrane in intact erythrocytes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):408–412. doi: 10.1073/pnas.79.2.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hsu L., Morrison M. A new variant of the anion transport protein in human erythrocytes. Biochemistry. 1985 Jun 18;24(13):3086–3090. doi: 10.1021/bi00334a003. [DOI] [PubMed] [Google Scholar]
  6. Jarolim P., Rubin H. L., Zhai S., Sahr K. E., Liu S. C., Mueller T. J., Palek J. Band 3 Memphis: a widespread polymorphism with abnormal electrophoretic mobility of erythrocyte band 3 protein caused by substitution AAG----GAG (Lys----Glu) in codon 56. Blood. 1992 Sep 15;80(6):1592–1598. [PubMed] [Google Scholar]
  7. Jennings M. L., Passow H. Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate. Biochim Biophys Acta. 1979 Jul 5;554(2):498–519. doi: 10.1016/0005-2736(79)90387-0. [DOI] [PubMed] [Google Scholar]
  8. Kampmann L., Lepke S., Fasold H., Fritzsch G., Passow H. The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid (H2DIDS). J Membr Biol. 1982;70(3):199–216. doi: 10.1007/BF01870563. [DOI] [PubMed] [Google Scholar]
  9. Landolt-Marticorena C., Casey J. R., Reithmeier R. A. Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein. Mol Membr Biol. 1995 Apr-Jun;12(2):173–182. doi: 10.3109/09687689509027505. [DOI] [PubMed] [Google Scholar]
  10. Levinson C., Corcoran R. J., Edwards E. H. Interaction of tritium-labeled H2DIDS (4,4'-diisothiocyano-1,2,diphenyl ethane-2,2'disulfonic acid) with the Ehrlich mouse ascites tumor cell. J Membr Biol. 1979 Mar 28;45(1-2):61–79. doi: 10.1007/BF01869295. [DOI] [PubMed] [Google Scholar]
  11. Mueller T. J., Morrison M. Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte. J Biol Chem. 1977 Oct 10;252(19):6573–6576. [PubMed] [Google Scholar]
  12. Okubo K., Kang D., Hamasaki N., Jennings M. L. Red blood cell band 3. Lysine 539 and lysine 851 react with the same H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid) molecule. J Biol Chem. 1994 Jan 21;269(3):1918–1926. [PubMed] [Google Scholar]
  13. Salhany J. M., Cordes K. A., Gaines E. D. Light-scattering measurements of hemoglobin binding to the erythrocyte membrane. Evidence for transmembrane effects related to a disulfonic stilbene binding to band 3. Biochemistry. 1980 Apr 1;19(7):1447–1454. doi: 10.1021/bi00548a028. [DOI] [PubMed] [Google Scholar]
  14. Salhany J. M., Cordes K. A., Schopfer L. M. Kinetics of conformational changes associated with inhibitor binding to the purified band 3 transporter. Direct observation of allosteric subunit interactions. Biochemistry. 1993 Jul 27;32(29):7413–7420. doi: 10.1021/bi00080a011. [DOI] [PubMed] [Google Scholar]
  15. Salhany J. M. Effect of chloride on the binding kinetics of various stilbenedisulfonates to band 3. Biochem Mol Biol Int. 1995 Aug;36(5):1067–1077. [PubMed] [Google Scholar]
  16. Salhany J. M., Schopfer L. M. Interactions between mutant and wild-type band 3 subunits in hereditary Southeast Asian ovalocytic red blood cell membranes. Biochemistry. 1996 Jan 9;35(1):251–257. doi: 10.1021/bi952411b. [DOI] [PubMed] [Google Scholar]
  17. Salhany J. M., Schopfer L. M., Kay M. M., Gamble D. N., Lawrence C. Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu). Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11844–11848. doi: 10.1073/pnas.92.25.11844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Salhany J. M., Sloan R. L., Cordes K. A. In situ cross-linking of human erythrocyte band 3 by bis(sulfosuccinimidyl)suberate. Evidence for ligand modulation of two alternate quaternary forms: covalent band 3 dimers and noncovalent tetramers formed by the association of two covalent dimers. J Biol Chem. 1990 Oct 15;265(29):17688–17693. [PubMed] [Google Scholar]
  19. Salhany J. M., Sloan R. L., Cordes K. A., Schopfer L. M. Kinetic evidence for ternary complex formation and allosteric interactions in chloride and stilbenedisulfonate binding to band 3. Biochemistry. 1994 Oct 4;33(39):11909–11916. doi: 10.1021/bi00205a029. [DOI] [PubMed] [Google Scholar]
  20. Schopfer L. M., Salhany J. M. Characterization of the stilbenedisulfonate binding site on band 3. Biochemistry. 1995 Jul 4;34(26):8320–8329. doi: 10.1021/bi00026a013. [DOI] [PubMed] [Google Scholar]
  21. Spring F. A., Bruce L. J., Anstee D. J., Tanner M. J. A red cell band 3 variant with altered stilbene disulphonate binding is associated with the Diego (Dia) blood group antigen. Biochem J. 1992 Dec 15;288(Pt 3):713–716. doi: 10.1042/bj2880713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tanner M. J., Martin P. G., High S. The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence. Biochem J. 1988 Dec 15;256(3):703–712. doi: 10.1042/bj2560703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Dort H. M., Low P. S., Cordes K. A., Schopfer L. M., Salhany J. M. Calorimetric evidence for allosteric subunit interactions associated with inhibitor binding to band 3 transporter. J Biol Chem. 1994 Jan 7;269(1):59–61. [PubMed] [Google Scholar]
  24. Wang D. N., Kühlbrandt W., Sarabia V. E., Reithmeier R. A. Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane. EMBO J. 1993 Jun;12(6):2233–2239. doi: 10.1002/j.1460-2075.1993.tb05876.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yannoukakos D., Vasseur C., Driancourt C., Blouquit Y., Delaunay J., Wajcman H., Bursaux E. Human erythrocyte band 3 polymorphism (band 3 Memphis): characterization of the structural modification (Lys 56----Glu) by protein chemistry methods. Blood. 1991 Aug 15;78(4):1117–1120. [PubMed] [Google Scholar]

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

RESOURCES