Affinity labelling and identification of the high-affinity choline carrier from synaptic membranes of Torpedo electromotor nerve terminals with [3H]choline mustard.
The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.
Rylett
R J
RJ
Abteilung Neurochemie, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, F.R.G.
D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002794:Choline / Q000031:analogs & derivatives* / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism*; D006426:Hemicholinium 3 / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D009466:Neuromuscular Blocking Agents; D012964:Sodium / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism*; D014101:Torpedo / Q000378:metabolism*
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D009466:Neuromuscular Blocking Agents; D006426:Hemicholinium 3; C013457:cyclocholine; D012964:Sodium; D002794:Choline