Abstract
Acetylcholine receptor from denervated rat leg muscle was purified 48,000-fold by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose. A control preparation containing only contaminants was made by performing a parallel purification in which alpha-bungarotoxin was added to the preparation prior to the cobrotoxin/Sepharose step. Comparison of the receptor was greater than 90% pure. Examination of the purified receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two major polypeptide chains with apparent weights of 45,000 and 51,000, along with minor components of 49,000, 56,000, 62,000, and 110,000. Polypeptides of the same molecular weight were found when purified acetylcholine receptor was radioiodinated and further purified by sucrose gradient sedimentation. Analysis of receptor purified from denervated muscles that had been incubated with [35S]methionine in organ culture showed that all of the identified polypeptide chains were radioactive, indicating that they had been synthesized de novo after denervation.
Highlights
Acetylcholine receptor from denervated rat leg muscle was purified 48,000-fold by affinity chromatography on concanavalin A/Sepharose and CobrotoxinlSepharose
Bungarotoxin was added to the preparation prior to the CobrotoxinlSepharose step
Purification of ACh Receptors-The purification scheme was designed to utilize the glycoprotein nature of muscle
Summary
Acetylcholine receptor from denervated rat leg muscle was purified 48,000-fold by affinity chromatography on concanavalin A/Sepharose and CobrotoxinlSepharose. Comparison of the receptor and control preparations showed that the receptor was greater than 90% pure. Examination of the purified receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two major polypeptide chains with apparent weights of 45,000 and 51,000, along with minor components of 49,000, 56,000, 62,000, and 110,000. Analysis of receptor purified from denervated muscles that had been incubated with [35SImethionine in organ culture showed that all of the identified polypeptide chains were radioactive, indicating that they had been synthesized de nouo after denervation. The ACh receptor has been isolated from muscle [2, 3] and from two related tissues, the electric organs of eel and of several species of marine ray. The electric tissues constitute a rich source of receptor, and receptors from both eel and Torpedo have been highly purified by a number of laboratories.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
