title: Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry.
abstract: The Amt family of high-affinity ammonium transporters is a family of integral membrane proteins that are found in archaea, bacteria, fungi, plants and animals. Furthermore, the family has recently been extended to humans with the recognition that both the erythroid and non-erythroid Rhesus proteins are also ammonium transporters. The Escherichia coli AmtB protein offers a good model system for the Amt family and in order to address questions relating to both its structure and function we have overproduced a histidine-tagged form of the protein (AmtB6H) and purified it to homogeneity. We examined the quaternary structure of AmtB6H (which is active in vivo) by SDS/PAGE, gel-filtration chromatography, dynamic light scattering and sedimentation ultracentrifugation. The protein was resistant to dissociation by SDS and behaved as a stable oligomer on SDS/PAGE. By equilibrium desorption chromatography we determined the mass ratio of dodecyl beta-D-maltoside to AmtB in the detergent-solubilized complex to be 1.03+/-0.03, and this allowed us to calculate, from analytical-ultracentrifugation data, that AmtB purifies as a trimer.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK.
authors.forename: Dan
authors.initials: D
authors.lastname: Blakey
authors.forename: Andrew
authors.initials: A
authors.lastname: Leech
authors.forename: Gavin H
authors.initials: GH
authors.lastname: Thomas
authors.forename: Graham
authors.initials: G
authors.lastname: Coutts
authors.forename: Kim
authors.initials: K
authors.lastname: Findlay
authors.forename: Mike
authors.initials: M
authors.lastname: Merrick
mesh_terms: D001483:Base Sequence; D001704:Biopolymers; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D008854:Microscopy, Electron; D010316:Particle Size; D014461:Ultracentrifugation
chemical_list: C111096:AmtB protein, E coli; D001704:Biopolymers; D027682:Cation Transport Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins