Isolation, immunochemical characterization and localization of the kainate sub-class of glutamate receptor from chick cerebellum.
The low-affinity kainate binding sites, present at high density in chick cerebellar membranes, were solubilized with Triton X-100 and purified 41-fold. The purified kainate binding sites, therein referred to as the kainate receptor, displayed the expected pharmacological specificity: domoate = kainate much greater than L-glutamate much greater than D-glutamate, quisqualate, N-methyl-D-aspartate. Analysed by SDS-PAGE under reducing conditions, a single polypeptide with a Mr = 49,000 was observed. Western blots of membranes prepared from different brain areas and animal species were analysed using a monoclonal antibody, named mAb IX-50, raised against the purified kainate receptor. The mAb IX-50 stained the 49,000 polypeptide in chick, goldfish and mammalian brain tissues indicating its conservation during evolution. The staining intensity correlated with the density of kainate binding sites. The mAb IX-50 stained also a 93,000 polypeptide but the latter did not copurify with the 49,000 polypeptide. The kainate binding activity was selectively immunoadsorbed on mAb IX-50 coupled to Sepharose which, upon elution, released a 49,000 polypeptide. The immunohistochemical localization of mAb IX-50 binding sites in the chick cerebellar molecular layer coincided with that of the kainate receptor. We conclude that the 49,000 polypeptide is part of the kainate receptor and carries the kainate recognition site.
Gregor
P
P
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
Eshhar
N
N
Ortega
A
A
Teichberg
V I
VI
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002531:Cerebellum / Q000032:analysis / Q000378:metabolism*; D002642:Chick Embryo; D002645:Chickens; D002850:Chromatography, Gel; D003902:Detergents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism*; D007150:Immunohistochemistry; D007608:Kainic Acid / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008970:Molecular Weight; D011863:Radioimmunoassay; D017470:Receptors, Glutamate; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D003902:Detergents; D005971:Glutamates; D007608:Kainic Acid; D008024:Ligands; D017470:Receptors, Glutamate; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter