I. Kasheverov et al., REVERSE-PHASE CHROMATOGRAPHY ISOLATION AND MALDI MASS-SPECTROMETRY OFTHE ACETYLCHOLINE-RECEPTOR SUBUNITS, Protein expression and purification, 12(2), 1998, pp. 226-232
Citations number
19
Categorie Soggetti
Biochemical Research Methods",Biology,"Biothechnology & Applied Migrobiology
A procedure for purifying the Torpedo californica nicotinic acetylchol
ine receptor subunits is proposed which involves preparative SDS-PAGE
followed by reverse-phase HPLC on a C-4 column in an acetonitrile-isop
ropanol system. By this method, the alpha-subunit can be completely se
parated 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-subuni
t of Na+,K+-ATPase comigrating with it on SDS-PAGE. The purity of all
acetylcholine receptor subunits thus obtained was verified by Edman de
gradation and MALDI mass-spectrometric analysis which could be perform
ed quite easily on the HPLC-purified samples. In general, we observed
a good correlation between the experimentally determined molecular mas
ses 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 sub
units into 1% octyl-beta-D-glucopyranoside generates samples suitable
for functional studies and enzymatic proteolysis or deglycosylation. (
C) 1998 Academic Press.