Characterization and partial purification of a chloride- and calcium-dependent glutamate-binding protein from rat brain.
A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.
Brose
N
N
Department of Neurochemistry, Max-Planck-Institute for Psychiatry, Martinsried, Federal Republic of Germany.
Halpain
S
S
Suchanek
C
C
Jahn
R
R
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002712:Chlorides / Q000502:physiology*; D004150:Dipeptidases / Q000378:metabolism; D043425:Glutamate Carboxypeptidase II; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D008081:Liposomes; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013570:Synaptic Membranes / Q000201:enzymology / Q000378:metabolism
D002712:Chlorides; D005971:Glutamates; D008081:Liposomes; D011510:Proteolipids; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D018698:Glutamic Acid; D004150:Dipeptidases; D043425:Glutamate Carboxypeptidase II; D002118:Calcium