Further Evidence for Multiple Forms of an N ‐Methyl‐ d ‐Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques

— Pretreatment with sulfhydryl‐reactive agents, such as N ‐ethylmaleimide and p ‐chloromercuriphenylsul‐fonic acid, invariably resulted in marked inhibition of the binding of dl ‐( E )‐2‐amino‐4‐[ 3 H]propyl‐5‐phosphono‐3‐pentenoic acid ([ 3 H]CGP 39653), a competitive antagonist at an N ‐methyl‐ d ‐aspartate (NMDA)‐sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X‐100, but did not significantly affect the binding of L‐[ 3 H]‐glutamic acid ([ 3 H]Glu), an endogenous agonist. The pre‐treatment was effective in reducing the binding of [ 3 H]‐CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl‐reactive agents also enhanced the potencies of NMDA agonists to displace [ 3 H]‐CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [ 3 H]Glu binding. The binding of both [ 3 H]CGP 39653 and [ 3 H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho‐lipase A 2 or C markedly inhibited [ 3 H]CGP 39653 binding without altering [ 3 H]Glu binding. Moreover, both phospho‐lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [ 3 H]CGP 39653 and [ 3 H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl‐reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [ 3 H]CGP 39653, with concomitant facilitation of that in an agonist‐preferring form by [ 3 H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.