A kainic acid receptor from frog brain purified using domoic acid affinity chromatography.
A kainic acid receptor was purified from Triton X-100/digitonin-solubilized frog brain membranes. The purification was carried out in two steps: ion exchange chromatography using DEAE-Sepharose CL-6B and affinity chromatography with domoic acid immobilized on Sepharose 4B. The specific binding activity of the affinity-purified receptor is 481-fold higher than that of the crude solubilized preparation and 1617-fold higher than that of the whole membrane fraction. Scatchard analyses of the affinity-purified receptor showed a curvilinear plot which fit a two-site model with dissociation constants of 5.5 and 34 nM and Bmax values of 1700 pmol/mg protein and 4400 pmol/mg protein for the high and low affinity components, respectively. The dissociation constants of the purified receptor are similar to those of the crude soluble preparation (4.8 and 39 nM). Inhibition constants for several kainic acid analogs were also similar for the purified and crude preparations. The active purified receptor migrated with a Mr = 570,000 on gel filtration analysis using Sepharose 6B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor showed a single broad band with silver stain, migrating with a Mr = 48,000.
Hampson
D R
DR
Laboratory of Neuro-otolaryngology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
Wenthold
R J
RJ
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003198:Computer Simulation; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008970:Molecular Weight; D011894:Rana pipiens; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification*
D007608:Kainic Acid; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter; C012301:domoic acid