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Kinetics of Carbamylcholine Binding to Membrane-Bound Acetylcholine Receptor Monitored by Fluorescence Changes of a Covalently Bound Probe

Dunn, Susan M. J. and Blanchard, Steven G. and Raftery, Michael A. (1980) Kinetics of Carbamylcholine Binding to Membrane-Bound Acetylcholine Receptor Monitored by Fluorescence Changes of a Covalently Bound Probe. Biochemistry, 19 (24). pp. 5645-5652. ISSN 0006-2960. doi:10.1021/bi00565a029.

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The fluorescent probe 5-(iodoacetamido)salicylic acid has been used to alkylate acetylcholine receptor enriched membrane fragments from Torpedo californica following their reduction with low concentrations of dithiothreitol. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-(iodoacetamido)salicylic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was abolished by preincubation of the membrane fragments with excess ɑ-bungarotoxin and was therefore specific for the acetylcholine receptor. Estimates of dissociation constants obtained from centrifugation experiments with radioactive ligand and from fluorescence titration data were in good agreement, showing that the observed fluorescence enhancement was an accurate reflection of receptor- carbamylcholine complex formation. The kinetics of carbamylcholine binding to labeled membrane fragments have been investigated over a wide range of ligand concentrations by using stopped-flow fluorescence techniques. The kinetic signal was complicated, and four distinct exponential phases were observed. A kinetic mechanism has been proposed to account for this behavior.

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Additional Information:© 1980 American Chemical Society. Received February 19, 1980; revised manuscript received July 3, 1980. Supported by US. Public Health Service Grant NS-10294 and by a grant from the Muscular Dystrophy Association of America. We thank Hsaio-Ping Moore for her help with the bromoacetylcholine labeling experiments. We are also grateful to John Racs for technical assistance and to Valerie Purvis for artwork and typing the manuscript.
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U.S. Public Health Service (USPHS)NS-10294
Muscular Dystrophy Association of AmericaUNSPECIFIED
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Caltech Church Laboratory of Chemical Biolog6176
Issue or Number:24
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Official Citation:Kinetics of carbamylcholine binding to membrane-bound acetylcholine receptor monitored by fluorescence changes of a covalently bound probe Susan M. J. Dunn, Steven G. Blanchard, and Michael A. Raftery Biochemistry 1980 19 (24), 5645-5652 DOI: 10.1021/bi00565a029
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67412
Deposited By: Ruth Sustaita
Deposited On:27 May 2016 18:58
Last Modified:11 Nov 2021 00:32

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