Kainate receptors in Xenopus central nervous system: solubilisation with n-octyl-beta-D-glucopyranoside.
[3H]Kainate binding to membrane homogenates and detergent extracts prepared from Xenopus central nervous system was evaluated in 50 mM Tris-citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with a KD of 54.4 nM to a large number of sites (Bmax = 27.8 pmol/mg of protein). Up to 80% of the total number of membrane-bound binding sites were solubilised using the nonionic detergent n-octyl-beta-D-glucopyranoside. Values for the KD of [3H]kainate for solubilised binding sites were 46.0 nM and 53.6 nM derived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similar Ki values in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min-1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor in Xenopus central nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest that Xenopus provides the most convenient model system yet investigated for biochemical analysis of kainate receptors.
Henley
J M
JM
MRC Molecular Neurobiology Unit, University of Cambridge Medical School, England.
Barnard
E A
EA
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D003902:Detergents; D005260:Female; D005960:Glucosides; D006027:Glycosides; D006054:Goldfish; D007608:Kainic Acid / Q000378:metabolism; D008297:Male; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012995:Solubility; D014981:Xenopus
D003902:Detergents; D005960:Glucosides; D006027:Glycosides; D007608:Kainic Acid; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter; C018619:octyl-beta-D-glucoside