Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase.
Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase.
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人红细胞乙酰胆碱酯酶 C 末端糖脂膜结合域中胺成分的鉴定。
DOI：
10.1021/bi00359a005
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Rosenberry,TL
中科院分区：
文献类型：
--
作者：
Haas,R;Brandt,PT;Knight,J;Rosenberry,TL
药理学系，凯斯西储大学医学院，克利夫兰，俄亥俄州44106接收日期：1985年10月25日;修订后的手册接收日期：1986年1月14日摘要：用饱和量的[14 C]甲醛和氰基硼氢化钠，通过还原性放射甲基化标记纯化的人红细胞乙酰胆碱酯酶。酸水解和自动氨基酸分析允许通过与放射性甲基化标准品共洗脱来鉴定放射性甲基化组分，并定量这些组分。观察到甲基化的N-末端氨基酸谷氨酸和精氨酸的水平分别为每70千道尔顿亚基0.66和0.34个残基，并且赖氨酸残基在其e-氨基上甲基化至每亚基7.40个残基的水平[哈斯，R.，& Rosenberry，T. L. 04 The Dog of the Woman（1985）148，154-162]，此外，每个亚基含有1.35个甲基化乙醇胺残基和0. 98位甲基化葡糖胺残基。木瓜蛋白酶消化切割成两个片段，一个酶活性的亲水性片段和一个小的疏水性片段，代表膜结合域的完整的酶。放射性甲基化的氨基酸定量保留在亲水性片段中，而甲基化的乙醇胺和葡萄糖胺仅限于疏水性结构域片段。该片段包括亚基的C-末端二肽。将通过手动Edman方法进行的肽测序与放射甲基化相结合，以证明疏水结构域片段的序列His-Gly-乙醇胺-Z。该序列中的乙醇胺残基与C-末端Gly的酰胺键连接，并且与Z中的乙醇胺残基明显不同，Z中的乙醇胺残基在完整的酶中易于发生放射甲基化。由于Z还包括葡糖胺和2摩尔脂肪酸[Roberts，W. L.，& Rosenberry，TL（1985）Biochem.Biophys.通信资源133，621-627]，我们得出结论，人红细胞乙酰胆碱酯酶的膜结合结构域是亚基C末端的共价连接糖脂。小鼠Thy-1糖蛋白和锥虫变体表面糖蛋白的膜结合结构域的类比进行了讨论。
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 Received October 25, 1985; Revised Manuscript Received January 14, 1986 abstract: Purified human erythrocyte acetylcholinesterase was labeled by reductive radiomethylationwith saturating amounts of [14C] formaldehyde and sodium cyanoborohydride. Acid hydrolysis and automated amino acid analysis permitted both identification of radiomethylated components by their coelution with radiomethylated standardsand quantitation of these components. The methylated N-terminal amino acids glutamate and arginine were observed at levels of 0.66 and 0.34 residues, respectively, per 70-kilodalton subunit, and lysine residues were methylated on their e-amino groups to a level of 7.40 residues per subunit [Haas, R., & Rosenberry, T. L.(1985) Anal. Biochem. 148, 154-162], In addition, each subunit contained 1.35 residues of methylated ethanolamine and0. 98 residue of methylated glucosamine. Papain digestion cleaved the intact enzyme into two fragments, an enzymatically active hydrophilic fragment and a small hydrophobic fragment that represented the membrane-binding domain. The radiomethylated amino acids were quantitatively retained inthe hydrophilic fragment, while the methylated ethanolamine and glucosamine were confined exclusively to the hydrophobic domain fragment. This fragment included the C-terminal dipeptide of the subunit. Peptide sequencing by manual Edman methods was combined with radiomethylation to demonstrate the sequence His-Gly-ethanolamine-Z for the hydrophobic domain fragment. The ethanolamine residue in this sequence is in amide linkage to the C-terminal Gly and is clearly distinct from the ethanolamine residues in Z which are susceptible to radiomethylation in the intact enzyme. Since Z also includes glucosamine and 2 mol of fatty acids [Roberts, W. L., & Rosenberry, TL (1985) Biochem. Biophys. Res. Commun. 133, 621-627], we conclude that the membrane-binding domain of human erythrocyte acetylcholinesterase is a covalently linked glycolipid at the C-termini of the subunits. Analogies to the membrane-binding domains of murine Thy-1 glycoprotein and trypanosome variant surface glycoproteins are discussed.