0000000000331219
AUTHOR
Uli Roth
Identification of Purine Binding Sites on Torpedo Acetylcholine Receptor
Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of…
Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.
Using 2,8,5'-[H-3]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allost…
Membrane Protein Subunit Fractionation by Means of Inverse Pore Gradient Elution Polyacrylamide Gel Electrophoresis
We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of…