Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1977 May;74(5):1851–1854. doi: 10.1073/pnas.74.5.1851

Microenvironment of the binding site in the lac carrier protein.

S Schuldiner, R Weil, D E Robertson, H R Kaback
PMCID: PMC431027  PMID: 325557

Abstract

Studies with a homologous series of (N-dansyl)aminoalkyl-1-thio-beta-D-galactopyranosides containing two to six methylene carbons bridging the galactosyl and dansyl ends of the molecules are described. The compounds were utilized in radioactive and nonradioactive form, and binding of each homologue to membrane vesicles isolated from Escherichia coli ML 308-225 was measured directly by flow dialysis in the presence of D-lactate. The results are compared with the D-lactate-induced fluorescence enhancement observed with each dansylgalactoside and with the ability of N-methylpicolinium perchlorate to quench the fluorescence of the bound homologues. The binding affinity of the lac carrier protein for the probes varies directly with the length of the alkyl linkage, and the same number of binding sites is observed with each homologue. In contrast, however, the increase in fluorescence observed upon binding varies dramatically as the alkyl chain is increased in length, with the fluorescence exhibiting maximal values at two and six methylene carbons and a minimum at four methylene carbons. Furthermore, quenching by N-methylpicolinium perchlorate exhibits an inverse relationship and maximum quenching is observed with the 4 carbon homologue. Possible reasons for this behavior are discussed.

Full text

PDF
1851

Selected References

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

  1. Chen R. F., Kernohan J. C. Combination of bovine carbonic anhydrase with a fluorescent sulfonamide. J Biol Chem. 1967 Dec 25;242(24):5813–5823. [PubMed] [Google Scholar]
  2. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  4. Ramos S., Schuldiner S., Kaback H. R. The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1892–1896. doi: 10.1073/pnas.73.6.1892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Reeves J. P., Shechter E., Weil R., Kaback H. R. Dansyl-galactoside, a fluorescent probe of active transport in bacterial membrane vesicles. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2722–2726. doi: 10.1073/pnas.70.10.2722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Rudnick G., Kaback H. R. Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with an impermeant azidophenylgalactoside. J Biol Chem. 1975 Sep 10;250(17):6847–6851. [PubMed] [Google Scholar]
  7. Rudnick G., Kaback H. R., Weil R. Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside. J Biol Chem. 1975 Feb 25;250(4):1371–1375. [PubMed] [Google Scholar]
  8. Rudnick G., Schildiner S., Kaback H. R. Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli. Biochemistry. 1976 Nov 16;15(23):5126–5131. doi: 10.1021/bi00668a028. [DOI] [PubMed] [Google Scholar]
  9. Schuldiner S., Kerwar G. K., Kaback H. R., Weil R. Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein. J Biol Chem. 1975 Feb 25;250(4):1361–1370. [PubMed] [Google Scholar]
  10. Schuldiner S., Kung H., Kaback H. R., Weil R. Differentiation between binding and transport of dansylgalactosides in Escherichia coli. J Biol Chem. 1975 May 25;250(10):3679–3682. [PubMed] [Google Scholar]
  11. Schuldiner S., Spencer R. D., Weber G., Weil R., Kaback H. R. Lifetime and rotational relaxation time of dansylgalactoside bound to the lac carrier protein. J Biol Chem. 1975 Dec 10;250(23):8893–8896. [PubMed] [Google Scholar]
  12. Schuldiner S., Weil R., Kaback H. R. Energy-dependent binding of dansylgalactoside to the lac carrier protein: direct binding measurements. Proc Natl Acad Sci U S A. 1976 Jan;73(1):109–112. doi: 10.1073/pnas.73.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Short S. A., Kaback H. R., Kohn L. D. Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J Biol Chem. 1975 Jun 10;250(11):4291–4296. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES