Kellaris K V, Ware D K, Smith S, Kyte J
Department of Chemistry, University of California at San Diego, La Jolla 92093.
Biochemistry. 1989 Apr 18;28(8):3469-82. doi: 10.1021/bi00434a048.
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.
通过在组织匀浆过程中用N - 乙基马来酰亚胺和碘乙酰胺对天然蛋白进行烷基化处理，以及在十二烷基硫酸盐溶液中使多肽展开时用N - 乙基马来酰亚胺对其进行烷基化处理，来评估加州电鳐电器官中乙酰胆碱受体各多肽链中游离半胱氨酸的数量。无法进行烷基化的半胱氨酸可解释为特定的胱氨酸，它们连接着各个多肽氨基酸序列中的位置。未还原的、烷基化的乙酰胆碱受体多肽用嗜热菌蛋白酶或胰蛋白酶消化。消化产物色谱图中的含胱氨酸肽段通过使用双金/汞电极进行电化学鉴定。已从这些消化产物中分离出三个嗜热菌蛋白酶肽段和三个胰蛋白酶肽段，并显示它们含有最初存在于天然蛋白中的完整胱氨酸。这些肽段中的大多数含有一个完整的分子内胱氨酸，连接着与α多肽中半胱氨酸128和142同源位置的两个半胱氨酸。乙酰胆碱受体各多肽链中的每一个这样的胱氨酸分别在至少一个肽段中被分离出来。这些含胱氨酸的肽段每一个还都含有葡糖胺。可以得出结论，在每个多肽各自同源位置出现的序列Asn - Cys - Thr/Ser中的每个天冬酰胺都被糖基化了，尽管在天冬酰胺与苏氨酸或丝氨酸之间存在一个胱氨酸。此外，乙酰胆碱受体α亚基中连接相邻半胱氨酸α192和α193的胱氨酸的存在[高，P. N.，& 卡林，A.（1986年）《生物化学杂志》261，8085 - 8088]已得到证实。
