Reverse-phase chromatography isolation and MALDI mass spectrometry of the acetylcholine receptor subunits.
A procedure for purifying the Torpedo californica nicotinic acetylcholine receptor subunits is proposed which involves preparative SDS-PAGE followed by reverse-phase HPLC on a C4 column in an acetonitrile-isopropanol system. By this method, the alpha-subunit can be completely separated from the 43-kDa protein which migrates very close to it on SDS-PAGE, and the delta-subunit can be isolated free from the beta-subunit of Na+, K(+)-ATPase comigrating with it on SDS-PAGE. The purity of all acetylcholine receptor subunits thus obtained was verified by Edman degradation and MALDI mass-spectrometric analysis which could be performed quite easily on the HPLC-purified samples. In general, we observed a good correlation between the experimentally determined molecular masses and those calculated from the amino acid sequences and when known, posttranslational modifications (glycosylation and phosphorylation) of individual receptor subunits. Transfer of the isolated receptor subunits into 1% octyl-beta-D-glucopyranoside generates samples suitable for functional studies and enzymatic proteolysis or deglycosylation.
Kasheverov
I
I
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
Utkin
Y
Y
Weise
C
C
Franke
P
P
Hucho
F
F
Tsetlin
V
V
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014101:Torpedo / Q000378:metabolism*
D006023:Glycoproteins; D011950:Receptors, Cholinergic; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase