The Escherichia coli AmtB protein is member of the ubiquitous Amt family of ammonium transporters. Using a variety of [14C]methylammonium-uptake assays in wild-type E. coli, together with amtB and glutamine synthetase (glnA) mutants, we have shown that the filtration method traditionally used to measure [14C]methylammonium uptake actually measures intracellular accumulation of methylglutamine and that the kinetic data deduced from such experiments refer to the activity of glutamine synthetase and not to AmtB. Furthermore, the marked difference between the Km values of glutamine synthetase calculated in vitro and those calculated in vivo from our data suggest that ammonium assimilation by glutamine synthetase is coupled to the function of AmtB. The use of a modified assay technique allows us to measure AmtB activity in vivo. In this way, we have examined the role that AmtB plays in ammonium/methylammonium transport, in the light of conflicting proposals with regard to both the mode of action of Amt proteins and their substrate, i.e. ammonia or ammonium. Our in vivo data suggest that AmtB acts as a slowly conducting channel for NH3 that is neither dependent on the membrane potential nor on ATP. Furthermore, studies on competition between ammonium and methylammonium suggest that AmtB has a binding site for NH4+ on the periplasmic face.
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Research Article|
August 09 2005
In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase
Arnaud Javelle;
Arnaud Javelle
*Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk NR4 7UH, U.K.
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Gavin Thomas;
Gavin Thomas
1
*Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk NR4 7UH, U.K.
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Anne-Marie Marini;
Anne-Marie Marini
†Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires, CP300 Rue des Pr. Jeener et Brachet, 12 6041 Gosselies, Belgium
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Reinhard Krämer;
Reinhard Krämer
‡Institute of Biochemistry, University of Köln, Zülpicher Strasse 47, 50674 Köln, Germany
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Mike Merrick
Mike Merrick
2
*Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk NR4 7UH, U.K.
2To whom correspondence should be addresed (email mike.merrick@bbsrc.ac.uk).
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Publisher: Portland Press Ltd
Received:
December 17 2004
Revision Received:
April 22 2005
Accepted:
May 05 2005
Accepted Manuscript online:
May 05 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 390 (1): 215–222.
Article history
Received:
December 17 2004
Revision Received:
April 22 2005
Accepted:
May 05 2005
Accepted Manuscript online:
May 05 2005
Citation
Arnaud Javelle, Gavin Thomas, Anne-Marie Marini, Reinhard Krämer, Mike Merrick; In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase. Biochem J 15 August 2005; 390 (1): 215–222. doi: https://doi.org/10.1042/BJ20042094
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