PARTIAL-PURIFICATION OF [H-3] GLUTAMATE-ASSOCIATING-PROTEINS WITH SENSITIVITY TO DISPLACEMENT BY N-METHYL-D-ASAARTATE FROM RAT-BRAIN

An attempt was made to solubilize and isolate [H-3]L-glutamic acid (Gl u) binding sensitive to displacement by N-methyl-D-aspartic acid (NMDA ) from rat brain. Brain synaptic membranes were solubilized by deoxych olic acid, followed by gel filtration with Sephadex G-25. In these tur bid supernatants, significant but fragile binding was detected with a variety of radioligands related to ionotropic subclasses of receptors for excitatory amino acids. These included ,11-dihydro-5H-dibenzo-[a,d ]cyclohepten-5,10-imine (MK-801), [H-3]glycine, [H-3]spermidine, [H-3] Glu, lpha-amino-3-hydroxy-5-methylisoxazole-4-propionic and [H-3]kaini c acids. Re-solubilization of turbid supernatants by Triton X-100 resu lted in detection of [H-3]Glu binding which was only stable for 24 h, with [H-3]MK-801 binding being entirely lost. In these clear preparati ons after re-solubilization, Glu was exclusively effective in complete ly displacing [H-3]Glu binding with other ligands being partially acti ve. Furthermore, [H-3]Glu binding displaceable by NMDA was eluted with 0.5 M KCl together with [H-3]Glu binding insensitive to NMDA on DEAE- Toyopearl column chromatography, while fractions eluted with 0.2 M KCl had NMDA-insensitive [H-3]Glu binding only. Chromatography on chelate (Zn)-Toyopearl resin resulted in elution of both NMDA-sensitive and N MDA-insensitive [H-3]Glu binding with 10 mM EDTA. High performance liq uid chromatography revealed that NMDA-sensitive [H-3]Glu binding was d etected at retention times of 10-20 min when eluted from an Asahipak E S-502N column with NaCl at linearly graded concentrations up to 0.5 M. In order to detect NMDA-sensitive [H-3]Glu binding, however, the whol e procedures needed to be completed within 24 h after re-solubilizatio n. Accordingly, the identity of the NMDA-sensitive [H-3]Glu binding pa rtially purified here is still unclear at present. The NMDA recognitio n domain could be more stable than the NMDA channel domain on the NMDA receptor ionophore complex under aqueous conditions.