Purification, biochemical characterization, binding activity, and selectivity of a glutamate binding protein from bovine brain.
A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D017830:Octoxynol; D011092:Polyethylene Glycols; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017470:Receptors, Glutamate; D013347:Subcellular Fractions / Q000378:metabolism
D000596:Amino Acids; D003902:Detergents; D005971:Glutamates; D011956:Receptors, Cell Surface; D017470:Receptors, Glutamate; D018698:Glutamic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol