Partial purification of [3H]glutamate-associating-proteins with sensitivity to displacement by N-methyl-D-aspartate from rat brain

Abstract

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