TY - JOUR
T1 - The affinity labeling of partially purified acetylcholine receptor from electric tissue of Electrophorus
AU - Karlin, A.
AU - Cowburn, D.
PY - 1973
Y1 - 1973
N2 - 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) benzyl trimethyl ammonium 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) benzyl trimethyl ammonium iodide reacted 5000 fold faster with the reduced receptor than did [14C]N ethylmaleimide. When purified receptor was labeled with [3H]4 (N maleimido) benzyl trimethyl ammonium 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 indicating a molecular weight of 40,000.
AB - 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) benzyl trimethyl ammonium 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) benzyl trimethyl ammonium iodide reacted 5000 fold faster with the reduced receptor than did [14C]N ethylmaleimide. When purified receptor was labeled with [3H]4 (N maleimido) benzyl trimethyl ammonium 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 indicating a molecular weight of 40,000.
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U2 - 10.1073/pnas.70.12.3636
DO - 10.1073/pnas.70.12.3636
M3 - Article
C2 - 4519650
AN - SCOPUS:0015722839
SN - 0027-8424
VL - 70
SP - 3636
EP - 3640
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 12 (I)
ER -