title: Further evidence for multiple forms of an N-methyl-D-aspartate recognition domain in rat brain using membrane binding techniques.
abstract: Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsulfonic acid, invariably resulted in marked inhibition of the binding of DL-(E)-2-amino-4-[3H]propyl-5-phosphono-3-pentenoic acid ([3H]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-[3H]-glutamic acid ([3H]Glu), an endogenous agonist. The pretreatment was effective in reducing the binding of [3H]-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 [3H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [3H]Glu binding. The binding of both [3H]CGP 39653 and [3H]Glu was similarly sensitive to pretreatment with four different proteases in Triton-treated membranes, whereas pretreatment with phospholipase A2 or C markedly inhibited [3H]CGP 39653 binding without altering [3H]Glu binding. Moreover, both phospholipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [3H]CGP 39653 and [3H]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 [3H]CGP 39653, with concomitant facilitation of that in an antagonist-preferring form by [3H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.
authors.affiliation: Department of Pharmacology, Setsunan University, Osaka, Japan.
authors.forename: P
authors.initials: P
authors.lastname: Zuo
authors.forename: K
authors.initials: K
authors.lastname: Ogita
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: D
authors.initials: D
authors.lastname: Han
authors.forename: Y
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D015763:2-Amino-5-phosphonovalerate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D008297:Male; D016202:N-Methylaspartate / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017470:Receptors, Glutamate / Q000187:drug effects / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*
chemical_list: D005971:Glutamates; D010879:Piperazines; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D013439:Sulfhydryl Reagents; C069052:CGP 39653; D018698:Glutamic Acid; D002731:4-Chloromercuribenzenesulfonate; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D015763:2-Amino-5-phosphonovalerate; C050592:3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide