Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.
Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D004926:Escherichia coli / Q000032:analysis*; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D009113:Muramidase; D011485:Protein Binding; D011955:Receptors, Drug; D013010:Sonication; D013104:Spheroplasts / Q000032:analysis
D000596:Amino Acids; D001426:Bacterial Proteins; D005971:Glutamates; D011955:Receptors, Drug; D009113:Muramidase