Biochemical and thermodynamic aspects of the binding of [3H]glycine to its strychnine-insensitive recognition site associated with the N-methyl-D-aspartate receptor complex.
The molecular mechanism of interaction between glycine and its strychnine-insensitive binding site linked to the N-methyl-D-aspartate receptor was investigated by examining on the one hand the thermodynamic properties of glycine binding, and, on the other hand, the effects of various functional group modifying agents on ligand binding. Raising the incubation temperature from 0 degrees to 37 degrees resulted in a consistent decrease of glycine binding affinity. Calculation of thermodynamic parameters from the corresponding Van't Hoff plot showed that the binding of glycine was mainly entropy-driven, the change in enthalpy contributing only little (25-30%) to the change in Gibbs free energy. Chemical modification with the sulfhydryl-directed agents p-hydroxy-mercuribenzoate and N-ethyl-maleimide showed free -SH groups to be critical for ligand binding to the receptor site. Furthermore, guanidino groups on arginyl residues, sensitive to 2,3-butanedione, were also found to participate in glycine binding. Both the -SH and the guanidino groups could be protected against their inactivation by co-incubation with glycine, indicating a direct involvement of these functional groups in the binding process. Dithiothreitol, a disulfide-reducing agent, likewise prevented [3H]glycine binding, suggesting that the glycine recognition site is stabilized by at least one disulfide bridge. It is concluded that the binding of glycine probably involves a strong ion-ion interaction between its carboxyl group and a positively charged guanidino group at the receptor site, resulting in a thermodynamically favorable increase in entropy by displacement of water molecules from the latter and a concomitant decrease in enthalpy. Furthermore, at least one free sulfhydryl group seems to participate in the binding process.
Schneider
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Sandoz Research Institute Berne Ltd, Switzerland.
Urwyler
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D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism* / Q000494:pharmacology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013331:Strychnine / Q000494:pharmacology*; D013696:Temperature; D013816:Thermodynamics; D014316:Tritium
D004220:Disulfides; D004852:Epoxy Compounds; D006902:Hydroxymercuribenzoates; D016194:Receptors, N-Methyl-D-Aspartate; D014316:Tritium; C030601:4-hydroxymercuribenzoate; C007366:diepoxybutane; D013331:Strychnine; D005033:Ethylmaleimide; D004229:Dithiothreitol; D005998:Glycine