Development of a micromethod to study the Na+-independent L-[3H]glutamic acid binding to rat striatal membranes. I. Biochemical and pharmacological characterization.
A micromethod was developed to measure the Na+-independent L-[3H]glutamic acid (Glu) binding to rat striatal membranes by using slightly purified membranes from very small tissue amounts, ranging from 0.2 to 0.5 mg wet tissue. The specific binding reached equilibrium in about 30 min incubation at 37 degrees C and was shown to be partly reversible. Scatchard's analysis of saturation data suggest the presence on striatal membranes of an apparent single homogeneous population of Na+-independent binding sites with Kd value 1.75 microM and Bmax 3.89 nmol/g protein. Hill's plot of these data was linear with slope not significantly different from unity, indicating the absence of cooperative interactions. Cl- and Ca2+ ions were shown to severely influence the L-[3H]Glu binding to striatal tissue. Maximal activating effects were obtained in the presence of both ions, although Cl- alone was shown to have a powerful stimulating action on the binding. Pharmacological studies suggested, however, the presence of at least two subpopulations of binding sites which bound quisqualic acid as well as ibotenic acid with differential affinities. L-aspartic acid and L-serine-O-sulfate were shown to be potent inhibitors of the L-[3H]Glu binding while DL-2-amino-4-phosphonobutyric acid (DL-APB) and glutamic acid diethylester (GDEE) competed with the binding but only at high concentrations. N-methyl-D-aspartic acid (NMDA), DL-2-amino-5-phosphonovaleric acid (DL-APV), D-alpha-aminoadipate (D-alpha AA) and kainic acid were shown not to significantly influence the binding of Glu to striatal membranes.
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D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003342:Corpus Striatum / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000502:physiology; D013696:Temperature
D000596:Amino Acids; D002712:Chlorides; D005971:Glutamates; D018698:Glutamic Acid; D012964:Sodium; D002118:Calcium