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. 1984 Apr;81(7):2223–2226. doi: 10.1073/pnas.81.7.2223

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

B E Peerce, E M Wright
PMCID: PMC345470  PMID: 6425830

Abstract

Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.

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Selected References

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

  1. Carilli C. T., Farley R. A., Perlman D. M., Cantley L. C. The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site. J Biol Chem. 1982 May 25;257(10):5601–5606. [PubMed] [Google Scholar]
  2. Hopfer U., Crowe T. D., Tandler B. Purification of brush border membrane by thiocyanate treatment. Anal Biochem. 1983 Jun;131(2):447–452. doi: 10.1016/0003-2697(83)90197-5. [DOI] [PubMed] [Google Scholar]
  3. Hosang M., Gibbs E. M., Diedrich D. F., Semenza G. Photoaffinity labeling and identification of (a component of) the small-intestinal Na+,D-glucose transporter using 4-azidophlorizin. FEBS Lett. 1981 Aug 3;130(2):244–248. doi: 10.1016/0014-5793(81)81130-1. [DOI] [PubMed] [Google Scholar]
  4. Jackson R. J., Mendlein J., Sachs G. Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase. Biochim Biophys Acta. 1983 May 26;731(1):9–15. doi: 10.1016/0005-2736(83)90391-7. [DOI] [PubMed] [Google Scholar]
  5. Karlish S. J. Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site. J Bioenerg Biomembr. 1980 Aug;12(3-4):111–136. doi: 10.1007/BF00744678. [DOI] [PubMed] [Google Scholar]
  6. Kaunitz J. D., Gunther R., Wright E. M. Involvement of multiple sodium ions in intestinal d-glucose transport. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2315–2318. doi: 10.1073/pnas.79.7.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kaunitz J. D., Wright E. M. Kinetics of sodium D-glucose cotransport in bovine intestinal brush border vesicles. J Membr Biol. 1984;79(1):41–51. doi: 10.1007/BF01868525. [DOI] [PubMed] [Google Scholar]
  8. Kessler M., Acuto O., Storelli C., Murer H., Müller M., Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta. 1978 Jan 4;506(1):136–154. doi: 10.1016/0005-2736(78)90440-6. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Mircheff A. K., Wright E. M. Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities. J Membr Biol. 1976 Sep 17;28(4):309–333. doi: 10.1007/BF01869703. [DOI] [PubMed] [Google Scholar]
  11. Nigg E. A., Cherry R. J. Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane. Biochemistry. 1979 Aug 7;18(16):3457–3465. doi: 10.1021/bi00583a004. [DOI] [PubMed] [Google Scholar]
  12. Pick U., Bassilian S. Modification of the ATP binding site of the Ca2+ -ATPase from sarcoplasmic reticulum by fluorescein isothiocyanate. FEBS Lett. 1981 Jan 12;123(1):127–130. doi: 10.1016/0014-5793(81)80035-x. [DOI] [PubMed] [Google Scholar]
  13. Pick U. Dynamic interconversions of phosphorylated and non-phosphorylated intermediates of the Ca-ATPase from sarcoplasmic reticulum followed in a fluorescein-labeled enzyme. FEBS Lett. 1981 Jan 12;123(1):131–136. doi: 10.1016/0014-5793(81)80036-1. [DOI] [PubMed] [Google Scholar]
  14. Schmidt U. M., Eddy B., Fraser C. M., Venter J. C., Semenza G. Isolation of (a subunit of) the Na+/D-glucose cotransporter(s) of rabbit intestinal brush border membranes using monoclonal antibodies. FEBS Lett. 1983 Sep 19;161(2):279–283. doi: 10.1016/0014-5793(83)81025-4. [DOI] [PubMed] [Google Scholar]
  15. Stevens B. R., Kaunitz J. D., Wright E. M. Intestinal transport of amino acids and sugars: advances using membrane vesicles. Annu Rev Physiol. 1984;46:417–433. doi: 10.1146/annurev.ph.46.030184.002221. [DOI] [PubMed] [Google Scholar]
  16. Weber J., Semenza G. Chemical modification of the small intestinal Na+/D-glucose cotransporter by amino group reagents. Evidence for a role of amino group(s) in the binding of the sugar. Biochim Biophys Acta. 1983 Jun 23;731(3):437–447. doi: 10.1016/0005-2736(83)90039-1. [DOI] [PubMed] [Google Scholar]

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