期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 387, 期 3, 页码 619-627出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.02.005
关键词
urea transporter; oligomeric state; ApUT; channel; 2D crystallization
资金
- Howard Hughes Medical Institute Funding Source: Medline
- NIDDK NIH HHS [R01 DK043955, DK43955] Funding Source: Medline
- NIGMS NIH HHS [P01 GM62580, R01 GM082927-02, R01 GM082927, P01 GM062580] Funding Source: Medline
Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured. urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family. (c) 2009 Elsevier Ltd. All rights reserved.
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