Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor.
Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor.
复制标题
评估游离半胱氨酸的数量以及从乙酰胆碱受体中分离和鉴定含胱氨酸的肽。
DOI：
10.1021/bi00434a048
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Kyte,J
中科院分区：
文献类型：
--
作者：
Kellaris,KV;Ware,DK;Smith,S;Kyte,J
加州大学圣迭戈分校化学系D-006，La Jolla，California 92093收到1988年9月19日；修订稿件收到1988年12月28日摘要：通过在组织匀浆过程中将天然蛋白与A-乙基马来酰亚胺和碘乙酰胺烷基化，并在多肽在十二烷基硫酸盐溶液中展开时与A-乙基马来酰亚胺烷基化，评估了来自加州鱼雷电子器官的每个乙酰胆碱受体多肽中游离半胱氨酸的数量。不能用于烷基化的半胱氨酸可以被解释为特定的半胱氨酸，连接在单个多肽的氨基酸序列中的位置。乙酰胆碱受体未还原的烷基化多肽用热裂解酶或胰酶消化。用金/汞双电极电化学法鉴定了消化图谱中含有半胱氨酸多肽。从这些消化液中分离出三个热裂解多肽和三个胰蛋白酶多肽，并证明它们含有原本存在于天然蛋白质中的完整半胱氨酸。这些多肽大多含有一个完整的分子内半胱氨酸，在与a多肽中的半胱氨酸128和142同源的位置连接两个半胱氨酸。从乙酰胆碱受体的每个多肽中分别分离出至少一个多肽中的每个半胱氨酸。这些含有半胱氨酸多肽中的每一个也含有氨基葡萄糖。可以得出结论，序列ASN-Cys-Thr/Ser中的每个天冬酰胺都是糖基化的，即使天冬酰胺和苏氨酸或丝氨酸之间有一个半胱氨酸，它出现在每个多肽中各自的同源位置。此外，在乙酰胆碱受体的α亚基[Kao，P.N.，&Karlin]中，证实了连接相邻半胱氨酸的半胱氨酸的存在。乙酰胆碱受体是一种跨膜糖蛋白，分子量约为250000(Noda et al.，1983c)，位于脊椎动物突触后膜上。它由四个亚基(a、/3、y和8)组成，它们的氨基酸序列彼此同源(Noda等人，
Department of Chemistry, D-006, University of California at San Diego, La Jolla, California 92093 Received September 19, 1988; Revised Manuscript Received December 28, 1988 abstract: 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 proteinwith A-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with A-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 thedigests 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 peptidescontained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the a 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, theexistence of the cystine connecting the adjacent cysteines,«192 and al93, in the a subunit of acetylcholine receptor [Kao, P. N., & Karlin, confirmed.. Acetylcholine receptor is a transmembrane glycoprotein of approximate molecular weight 250000 (Noda et al., 1983c) located in the postsynaptic membranes of vertebrates. It is made up of four subunits (a,/3, y, and 8) that are homologous to each other in their amino acid sequences (Noda et al.,