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Biomedical subjects

A Karlin

Publications and source records attributed to A Karlin.

At least 91 records · Page 5Linked to original sources
PubMed1973

The affinity-labeling of partially purified acetylcholine receptor from electric tissue of Electrophorus.

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-(3)H]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, [(3)H]4-(N-maleimido)-benzyltrimethylammonium iodide reacted 5000-fold faster with the reduced receptor than did [(14)C]N-ethylmaleimide. When purified receptor was labeled with [(3)H]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 (3)H activity; its mobility indicated a molecular weight of 40,000.

Alkylating Agents↗
PubMed1972

Affinity labeling of the acetylcholine receptor in the electroplax: electrophoretic separtion in sodium dodecyl sulfate.

Electroplax, single cells dissected from electric tissue of Electrophorus, are labeled in a two-step procedure: reduction by dithiothreitol followed by alkylation by the affinity label 4-(N-maleimido)-alpha-benzyltri-[methyl-(3)H]methylammonium iodide, either alone or in combination with [2,3-(14)C]N-ethylmaleimide. Electrophoresis in sodium dodecyl sulfate on polyacrylamide gel of an extract, prepared with this detergent, of single-labeled or of double-labeled cells results in a major peak of (3)H activity, with a mobility corresponding to a polypeptide of molecular weight 42,000. In addition, in the double-labeled samples, there is a unique peak in the ratio of (3)H to (14)C that is coincident with the (3)H peak. The electrophoretic patterns of extracts of cells in which affinity alkylation of the reduced receptor has been suppressed by dithiobischoline, an affinity oxidizing agent, by cobratoxin, an irreversible ligand, or by hexamethonium, a reversible ligand, show a considerably diminished peak of (3)H activity in the region of molecular weight 42,000. This is the predominant difference between the electrophoretic patterns of extracts of unprotected and of protected cells. Furthermore, extracts of cells protected with dithiobischoline before labeling with both tritiated affinity label and [(14)C]N-ethylmaleimide do not show the peak in the (3)H to (14)C ratio seen in the absence of protection. Thus, by several diverse criteria, the peak of (3)H activity corresponding to a molecular weight of 42,000 contains affinity-labeled acetylcholine receptor or receptor subunit.

Acrylamides↗
PubMed1969

Acetylcholine receptor: covalent attachment of depolarizing groups at the active site.

Following reduction of the acetylcholine receptor in the electroplax with dithiothreitol, the quaternary ammonium compounds bromoacetylcholine bromide and the p-nitrophenyl ester of (p-carboxyphenyl) trimethylammonium iodide react near the active site probably with a sulfhydryl group. The covalently attached quaternary ammonium moieties additionally interact with the active site noncovalently to activate the receptor and cause depolarization of the cell.

Acetylcholine↗