期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 105, 期 26, 页码 8896-8901出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0803577105
关键词
lactose permease; membrane transporters; pH titrations; proton translocation; substrate affinity
资金
- NIDDK NIH HHS [R01 DK051131-13, R01 DK051131-11, R01 DK069463-02, R01 DK069463-04, R56 DK069463, R01 DK069463, R01 DK051131-06, R01 DK051131, R01 DK051131-10, R01 DK051131-08, R56 DK051131, R01 DK051131-07, R01 DK069463-03, R01 DK051131-09, R01 DK051131-05, R01 DK051131-12, DK069463, R56 DK069463-05A1, R01 DK051131-04, R01 DK069463-01, DK51131, R01 DK051131-14] Funding Source: Medline
- NIGMS NIH HHS [GM074929, U54 GM074929-020005, U54 GM074929, U54 GM074929-010005, P50 GM073210, U54 GM074929-040005, U54 GM074929-050005, U54 GM074929-030005, GM073210] Funding Source: Medline
The effect of bulk-phase pH on the apparent affinity (K-d(app)) of purified wild-type lactose permease (LacY) for sugars was studied. K-d(app) values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K-d(app) for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pK(a) of approximate to 10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H+ symport in either direction across the membrane.
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