Acetylcholine receptors from normal human muscle: Concentration, purification, and use in radioreceptor assays for autoantibodies
✍ Scribed by James T. Wu; Joseph A. Knight; Terry G. Miya; Cory Bringhurst
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 634 KB
- Volume
- 5
- Category
- Article
- ISSN
- 0887-8013
No coin nor oath required. For personal study only.
✦ Synopsis
We measured the concentration of acetylcholine receptor (AChR) in various normal human muscles, obtained at autopsy, in order to find a more reliable and convenient source than the amputated leg muscle from diabetic patients for the isolation of AChR. We found that human calf muscle contains t h e highest concentration of AChR, approaching the concentration of receptor required for the radioreceptor assay for autoantibodies in myasthenia gravis (MG). Because the amount of contaminant proteins in the receptor preparations affect the sensitivity and precision of the assay, various chromatographic tech-
Key words:
niques were tested to improve the purity of the receptor preparation. We found that both G-100 Sephadex and DEAE Sephacel chromatographies were effective in removing contaminant proteins. OEAE Sephacel chromatography is particularly useful because the procedure provides higher recovery of receptor. Against the same pool of autoantibodies from patients with MG, AChRs from diabetic and normal leg muscle exhibit similar affinities. We conclude that AChR from normal human calf muscle can be used in radioreceptor assays for measuring autoantibodies in patients with myasthenia gravis. isolation, DEAE-Sephacel chromatography, gel filtration chromatography, G-1 00, Con A Sepharose chromatography, nonspecific binding
Methods
Sephadex G-100, DEAE Sephacel, and Blue Dextran 2000 were purchased from Pharmacia (Piscataway, NJ). Trizma (or phosphate), sodium thiocyanate, Triton X-100, methyl-a-Dglucopyranoside, a-methyl-D-glucoside, EDTA, EGTA, iodoacetamide, and phenylmethanesulfonyl fluoride (PMSF)