Purification and properties of a periplasmic glutamate-aspartate binding protein from Escherichia coli K12 strain W3092.
A protein which binds both glutamate (K-D = 0.8 muM) and aspartate (K-D = 1.2 muM) has been purified to homogeneity (290-fold) from the periplasmic fraction released from Escherichia coli W3092 by the cold osmotic shock procedure. The apparent molecular weight of the glutamate-aspartate binding protein is approximately 31,000 as judged by gel electrophoresis, gel filtration, and sedimentation equilibrium centrifugation; and the protein has a pI of 9.69. This protein contains 2 half-cystine residues and is dependent on a dithiothreitol-sensitive component for renaturation to an active conformation following urea or guanidine treatment. Of the natural amino acids only the L isomers of glutamate, aspartate, glutamine, asparagine, and alanine were inhibitors of either [C]glutamate or [14C]aspartate binding and the inhibitions were competitive. Only one binding site is indicated per molecule of protein. Antibody prepared against the glutamate-asparate binding protein does not cross-react with purified glutamine binding protein or any other component of osmotic shock fluid. The antibody does cross-react with osmotic shock fluids obtained from E. coli strains B and W and Salmonella typhimurium OT2. The glutamate-aspartate binding protein-antibody complex does not bind either glutamate or aspartate. The protein may be similar to the glutamate binding activity detected in the periplasmic fraction released from E. coli strain B (Miner, K.M., and Frank, L. (1974) J. Bacteriol. 117, 1093-1098) and strain K12 CS (Barash, H., and Halpern, Y.S. (1971) Biochem. Biophys. Res. Commun. 45, 681-688). This protein appears to function in the transport of glutamate by E. coli strain W cultured in minimal medium with succinate as the carbon source (Willis, R.C., and Furlong, C.E. (1975) J. Biol. Chem. 250, 2581-2586.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D011817:Rabbits / Q000276:immunology; D011955:Receptors, Drug; D013386:Succinates / Q000494:pharmacology
D000596:Amino Acids; D001426:Bacterial Proteins; D005971:Glutamates; D011955:Receptors, Drug; D013386:Succinates; D001224:Aspartic Acid