Solubilization and characterization of kainate receptors from goldfish brain.
The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.
Henley
J M
JM
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
Oswald
R E
RE
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D006054:Goldfish; D007608:Kainic Acid / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012016:Reference Values; D012995:Solubility
D007608:Kainic Acid; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter