title: Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli.
abstract: The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [gamma-32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system.
mesh_terms: D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000032:analysis; D001120:Arginine / Q000378:metabolism; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009952:Ornithine / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D010766:Phosphorylation; D013379:Substrate Specificity
chemical_list: D026905:Amino Acid Transport Systems; D000596:Amino Acids; D002352:Carrier Proteins; D033902:Periplasmic Binding Proteins; C035727:arginine-ornithine periplasmic transport protein, bacteria; D001120:Arginine; D009952:Ornithine