Abstract
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.
Highlights
Cation of a novel ATPasefrom a bacterialstrain which hyperproduces the mglA gene product of S. typhimurium.The purified protein shows a single band when analyzed by electrophoresisinnondenaturing polyacrylamide gels, and the ATPase activity coincides with the protein peak during the last steps of the purification procedure
Purification of the MglA ATPase-The MglA protein was produced ina mgl mutant of E . coli transformed with a pBR322 plasmid containing the mglA and mglB genes of S. typhimurium
51,000, 38,000, and 15,000 daltons on sodium dodecyl sulfate polyacrylamide gels (Fig. 1B). These molecular masses are similar to those of the two forms (51,000 and 38,000 daltons) of the MglA protein determined from molecular cloning experiments[5],and the 15,000-dalton band is probably the second proteolytic product of the 51,000-daltonprotein (these polypeptides and ATPase activity were not observed when a similar purification procedure was made from strain LA 5709 pHG16 which does not hyperproduce MglA)
Summary
Purification of the MglA ATPase-The MglA protein was produced ina mgl mutant of E . coli transformed with a pBR322 plasmid containing the mglA and mglB genes of S. typhimurium. 51,000, 38,000, and 15,000 daltons on sodium dodecyl sulfate polyacrylamide gels (Fig. 1B) These molecular masses are similar to those of the two forms (51,000 and 38,000 daltons) of the MglA protein determined from molecular cloning experiments[5],and the 15,000-dalton band is probably the second proteolytic product of the 51,000-daltonprotein (these polypeptides and ATPase activity were not observed when a similar purification procedure was made from strain LA 5709 pHG16 which does not hyperproduce MglA (notshown)). The peak of ATPase activity eluted from the hydroxylapatite column (and from a subsequent gel permeation column (see below)) coincides with the peak of MglAprotein (Fig. 2). This suggests that the ATPase described here is the MglA gene product. Antibodies raised against the purified MglA protein specificallryecognize three different polypeptides (molecular masses, 51,000, 38,000, and 15,000 daltons) in crude extracts from a MglA producing strain (lane1 ) ;these polypeptides are not apparent in control extractsfrom a strainwhich doesnot
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