Abstract
A Triton X-100 extract of electric tissue was subjected to a single step affinity chromatography using either of two affinity gels: [N-(6-aminocaproyl)-p-aminobenzyl]trimethylammonium bromide or methyl[(6-aminocaproyl-6′-aminocaproyl)-3-amino]pyridinium bromide attached to Sepharose 4B. Specific elution of the acetylcholine receptor-I (AcChR-I) with low concentration of a bis-quaternary agonist, 3,3′-bis[α-(trimethylammonium)methyl]-azobenzene bromide (Bis-Q), gave a 35% yield of toxin-binding components in the crude extract. The purified AcChR-I readily underwent aggregation, which appeared to arise from the oxidation of titratable free sulfhydryl on the protein. The protein was characterized by the binding capacities for [125I]α-bungarotoxin (α-Bgt), [3H]acetylcholine, and [14C]Bis-Q; the ratio of these capacities were approximately 2:1:2, respectively, with 5-6:5 nmole of α-Bgt sites per mg of protein. Analysis by sodium dodecyl sulfate gel electrophoresis of the disulfide-reduced and nonreduced polypeptide components indicated that a 41,500 dalton species was the major subunit component of AcChR-I. The binding of [14C]Bis-Q with a Triton X-100 crude extract showed sites with both high and low dissociation constants, whereas purified AcChR-I contained only high-affinity sites. A biphasic double-reciprocal plot and a Hill coefficient of 0.7 suggested negative cooperativity in the binding of Bis-Q with the purified AcChR-I.
Keywords: affinity chromatography, membrane-bound protein, subunit structure, sulfhydryl titration, equilibrium dialysis
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