Reconstitution of the binding protein-dependent galactose transport of Salmonella typhimurium in proteoliposomes.
Binding protein-dependent transport systems mediate the accumulation of diverse substrates in bacteria. The binding protein-dependent galactose transport of Salmonella typhimurium has been reconstituted in proteoliposomes. The proteoliposomes were made with proteins solubilized and renatured from inclusion bodies produced by a bacterial strain containing a plasmid with the mgl (methylgalactose permease) operon of Salmonella typhimurium. Galactose transport is dependent both on the addition of the purified galactose binding protein to the transport assay, and on ATP. The interaction between the liganded galactose binding protein and proteoliposomes displays Michaelis type kinetics with a Km of around 15 microM. Galactose transport is coupled to ATP hydrolysis with a stoichiometry (ATP/galactose) of 2.5:1. Galactose transport in proteoliposomes is not significantly inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone, but is inhibited by 0.5 mM vanadate. The present reconstitution of galactose transport in proteoliposomes suggests that the MglA, MglC and MglE proteins have been solubilized and renatured in an active form from the inclusion bodies of the mgl hyperproducing strain.
Richarme
G
G
Biochimie Génétique, Institut Jacques Monod, Université Paris, France.
el Yaagoubi
A
A
Kohiyama
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D001693:Biological Transport, Active; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000378:metabolism*; D012995:Solubility
D001425:Bacterial Outer Membrane Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011510:Proteolipids; C023550:galactose-binding protein; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D005690:Galactose