The MglA component of the binding protein-dependent galactose transport system of Salmonella typhimurium is a galactose-stimulated ATPase.
Binding protein-dependent transport systems mediate the accumulation of several ions, sugars, amino acids, and peptides in Gram-negative bacteria by using the energy of ATP hydrolysis and belong to a superfamily of membrane proteins which extends to eukaryotic cells and includes the multidrug resistance P-glycoprotein and the cystic fibrosis transmembrane conductance regulator. The binding protein-dependent galactose transport system of Salmonella typhimurium comprises four proteins which have been characterized previously by molecular cloning experiments (51,000-dalton MglA protein, with a stable proteolytic product of 38,000 daltons, 33,000-dalton MglB protein, 29,000-dalton MglC protein, 21,000-dalton MglE protein). By using a MglA hyperproducing strain, we have purified a galactose-stimulated ATPase which shows a single band in polyacrylamide gels under nondenaturing conditions and shows three bands at 51,000, 38,000, and 15,000 daltons on sodium dodecyl sulfate-polyacrylamide gels (our results suggest that the bands at 38,000 and 15,000 daltons represent proteolytic products of the 51,000-dalton protein). The ATPase activity coincides with the purified protein during the two last chromatographic steps of the purification procedure, and it cannot be isolated from a strain which does not contain the mglA gene. The MglA ATPase is stimulated 3-fold by galactose and hydrolyzes ATP to ADP and Pi (Km ATP = 60 microM, Ka galactose = 0.3 mM, Vmax = 140 nmol/min/mg of protein). The gamma-phosphate of ATP is transferred neither to galactose nor to the protein itself. Vanadate, N-ethylmaleimide and 5-methoxyindole-2-carboxylic acid, a specific inhibitor of binding protein-dependent transport systems, inhibit the MglA ATPase.
Richarme
G
G
Institut J. Monod, Université Paris 7, France.
el Yaagoubi
A
A
Kohiyama
M
M
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002845:Chromatography; D002846:Chromatography, Affinity; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism*; D005798:Genes, Bacterial; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D006882:Hydroxyapatites; C071728:MglA protein, Myxococcus xanthus; D000255:Adenosine Triphosphate; D017886:Durapatite; D000251:Adenosine Triphosphatases; D005690:Galactose