Binding of l-[(3)H]glutamate to repeatedly frozen-thawed rat brain membranes

l-[(3)H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2-4 degrees C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, K(D) = 697 nM; maximal binding capacity, B(max) = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (K(D1) = 26 nM, B(max1) = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (K(D2) = 662 nM, B(max2) = 10.5 pmol/mg protein). l-[(3)H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors.