Covalent modification of a critical sulfhydryl group in the acetylcholine receptor: cysteine-222 of the alpha-subunit.
Chemical modification of the Torpedo californica acetylcholine receptor (AcChR) by the fluorescent agent N-(1-pyrenyl)maleimide (PM) under nonreducing conditions resulted in the labeling of cysteine residues in all subunits and marked inhibition of the AcChR ion channel opening [Clarke, J. H., & Martinez-Carrion, M. (1986) J. Biol. Chem. 261, 10063-10072]. The PM alkylation kinetics are not affected by the presence of agonists or a competitive antagonist. The PM-labeled alpha-subunit has been purified and digested with both CNBr and trypsin. The resulting fragments from both cleavages were fractionated by high-performance liquid chromatography. The amino acid analysis and sequencing data of the PM-labeled peptides identified cysteine-222 as the only residue labeled by PM on the alpha-subunit primary structure. Cysteine-222 is located in the middle of a hydrophobic domain designated M1, which contains a homologous class of cysteines (Cys-241 in the aligned sequences) conserved in the four subunits of the AcChR. Because of its reactivity and fluorescent properties of the bound probe, alpha Cys-222 seems to be free sulfhydryl group accessible through a hydrophobic pocket, and these properties should be incorporated into proposed folding models for the alpha-subunit.
Marquez
J
J
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110.
Iriarte
A
A
Martinez-Carrion
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014101:Torpedo
D000596:Amino Acids; D005456:Fluorescent Dyes; D008301:Maleimides; D011950:Receptors, Cholinergic; D013438:Sulfhydryl Compounds; C003944:N-(3-pyrene)maleimide; D003545:Cysteine