L-Aspartate binding sites in rat cerebellum: a comparison of the binding of L-[3H]aspartate and L-[3H]glutamate to synaptic membranes.
The binding of L-[3H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (Kd = 874 nM and Bmax = 44 pmol/mg protein), which displayed no cooperativity (Hill coefficient approx. = 1). Specific [3H]aspartate was readily and reversibly displayed by unlabelled L-aspartate (the D-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [3H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists, DL-alpha-aminoadipate, DL-alpha-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-D-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate. While freezing or cold storage of membranes resulted in diminished [3H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site. Differential effects of various cations on [3H]aspartate binding were observed--monovalent cations reduced, while divalent cations enhanced L-[3H]aspartate binding.
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002412:Cations; D002531:Cerebellum / Q000378:metabolism*; D005260:Female; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism*; D013696:Temperature; D014316:Tritium
D002412:Cations; D005971:Glutamates; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid