Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor.
The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.
Kellaris
K V
KV
Department of Chemistry, University of California at San Diego, La Jolla 92093.
Ware
D K
DK
Smith
S
S
Kyte
J
J
D000595:Amino Acid Sequence; D000818:Animals; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis; D003553:Cystine / Q000032:analysis; D004557:Electric Organ / Q000032:analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D014101:Torpedo
D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D003553:Cystine; D003545:Cysteine; D003488:Cyanogen Bromide