Characterization and Partial Purification of the Acetylcholine Receptor from Torpedo Electroplax

Binding of acetylcholine in the concentration range 1 nM-1 μM was measured by equilibrium dialysis to a particulate preparation ofTorpedo electroplax, without or with prior treatment of the tissue with one of three chemical modifying reagents. Significant reduction in binding of acetylcholine resulted after treatment with 1,4-dithiothreitol,p -chloromercuribenzoate, orp -(trimethylammonium)-benzenediazonium fluoroborate. Partial reversal of the reduction in binding occurred when dialysis was performed in the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) or potassium ferricyanide (in the case of treatment with dithiothreitol), and 2-mercaptoethanol (in the case of treatment withp -chloromercuribenzoate). It is concluded that the functional acetylcholine-receptor macromolecule ofTorpedo electroplax has disulfide bond(s), sulfhydryl group(s), and one or more of the amino acids vulnerable to diazotization byp -(trimethylammonium)-benzenediazonium fluoroborate. This, plus the effect of phospholipase C (EC 3.1.4.3) in elimination of detectable binding of acetylcholine after electrofocusing, is additional evidence that the functional acetylcholine receptor is a phospholipoprotein or a phospholipid-protein complex, which has a low isoelectric point of 4.5 ± 0.2, yet is denatured by exposure to low pH for 24 hr. Due to this adverse effect, recovery of binding of acetylcholine after electrofocusing, as detected by equilibrium dialysis or ultrafiltration, is only 23% and, so far, only 6.3-fold purification of functional acetylcholine receptors by this technique is possible.Three or two forms of acetylcholinesterase (EC 3.1.1.7), whose peaks have isoelectric points ranging from 4.3 to 7.2, appear after electrofocusing ofTorpedo extracted with 1% Triton X-100 or Lubrol, respectively. The major peak in either preparation has an isoelectric point of 5. Although the peaks of the functional acetylcholine receptors and of acetylcholinesterase ofTorpedo electroplax are separable by electrofocusing, it has not been possible to isolate fractions that contain functional receptors but that are free of acetylcholinesterase. The opposite is possible.