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标题：The Affinity-Labeling of Partially Purified Acetylcholine Receptor from Electric Tissue of Electrophorus
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作者：Arthur Karlin ; David Cowburn
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1973
卷号：70
期号：12
页码：3636-3640
DOI：10.1073/pnas.70.12.3636
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The receptor for acetylcholine was partially purified by affinity chromatography of an extract in Triton X-100 of membrane fragments from electric tissue. The receptor was assayed, after its reduction with dithiothreitol, by reaction with the affinity-alkylating agent, [methyl-3H]4-(N-maleimido)-benzyltrimethylammonium iodide. Alternative labeling procedures, one useful for routine assay of picomole quantities of receptor and the other for labeling larger quantities of receptor, are described. The purified receptor specifically incorporated about 3 nmol of label per mg of protein. This incorporation was blocked by pretreatment of the receptor with Naja naja siamensis neurotoxin. The rate of the affinity reaction was similar to that found with membrane fragments and with intact electroplax. Furthermore, as in intact electroplax, [3H]4-(N-maleimido)-benzyltrimethylammonium iodide reacted 5000-fold faster with the reduced receptor than did [14C]N-ethylmaleimide. When purified receptor was labeled with [3H]4-(N-maleimido)-benzyltrimethylammonium iodide and subjected to electrophoresis on polyacrylamide gels in dodecyl sulfate and dithiothreitol, three major protein bands were observed. Only one of these, however, contained 3H activity; its mobility indicated a molecular weight of 40,000.
关键词：affinity chromatography ; 4-( N -maleimido)-benzyltrimethylammonium ; labeling kinetics ; membrane proteins ; subunit molecular weight