Purification and Localization of Kainate Binding Protein in Pigeon Cerebellum

Abstract

Excitatory amino acid (EAA) receptors are pharmacologically well characterized (Watkins and Evans, 1981; Foster and Fagg, 1984; Cotman et al., 1987; Mayer and Westbrook, 1987; Watkins and Olvermann, 1987), but virtually unknown at the molecular level. Kainic acid (KA), a heterocyclic L-glutamate analogue, defines as agonist a certain type of these receptors. The results of binding studies have indicated that brain tissues of lower vertebrates are relatively rich in kainate binding sites (Henke and Cuénod, 1980; London et al., 1980) and, thus, might serve as sources for molecules related to EAA receptors. In particular, Henke and collaborators (Henke et al., 1981) have described a high abundance (B_max =118 pmol/mg protein) of kainic acid binding sites with relatively low affinity (K_d = 330 nM) in the pigeon cerebellum and have localized them autoradiographically with tritiated kainate in the molecular and Purkinje cell layers. From this tissue, kainate binding activity has been solubilized by means of the non-ionic detergent Triton X-100 (Dilber et al., 1983). - In the present contribution, we report on a monoclonal antibody (mAb) used to purify a kainite binding protein (KBP) from pigeon cerebellum and to localize it immunohisto-chemically. Furthermore, we describe the characteristics of polyclonal antibodies to purified KBP in immunohistochemical and immunoblotting experiments. Part of this work has been published previously (Klein et al., 1988 a,b).