| 4525321 |
Chang HW: Purification and characterization of acetylcholine receptor-I from Electrophorus electricus. Proc Natl Acad Sci U S A. 1974 May;71(5):2113-7.
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 [alpha-(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 [(125) I] alpha-bungarotoxin (alpha-Bgt), [(3) H] acetylcholine, and [(14) C] Bis-Q; the ratio of these capacities were approximately 2:1:2, respectively, with 5-6:5 nmole of alpha-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 [(14) C] 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. |
32(0,1,1,2) |