Abstract
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.
Highlights
A kainic acid receptor was purified from Triton X100/digitonin-solubilizedfrog brain membranes
We have recently reported the solubilization of an active KA binding protein from rat brain (Hampson et al, 1987b)
Gel Filtration Chromatography-Samples (5 ml) of the crude solubilized or purified preparations were applied to a column (1.5 X 90 cm) of Sepharose 6B equilibrated with 50 mM Tris citrate, pH 7.0, containing 0.1% Triton X-100, 5% glycerol, and 100 mM NaC1
Summary
Vol 263, No 5, Issue of February 15, pp. 2500-2505,1988 Printed in U.S.A. ILnastbioturatetosmifoHf eNaeltuhr,oB-oettohleasr“dvawI ,oMloamry.laNnadti2o0n8a9l 2Instit ute of Neurological and. The development of selective pharmacological agents has allowed the electrophysiological characterization of neuronal receptors for excitatory amino acids. We have recently reported the solubilization of an active KA binding protein from rat brain (Hampson et al, 1987b) While this representsthe initial steptoward the characterization of the KA receptor, the purification of this receptor is hampered by its low concentration in mammalian brain and the lack of an efficient method of purification. It has been reported that brains of several species have much higher levels of KA receptor than does rat brain, with frog brain being among the highest (London et al, 1980).In the present study we have used frog brain as a source for the purification of a KA receptor This purification was facilitated by our development of a domoic acidaffinity column
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