Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase.
Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase.
复制标题
人红细胞乙酰胆碱酯酶疏水膜结合域的选择性放射性标记和分离。
DOI：
10.1021/bi00359a004
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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：
Roberts,WL;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Mandarin pt接收日期：1986年1月14日摘要：纯化的人红细胞乙酰胆碱酯酶的疏水性膜结合结构域用光活化试剂3-（三氟甲基）-3-（w-[125 I]碘苯基）二氮丙啶标记。放射性标记掺入时，酶制备在无洗涤剂的聚集体中，在洗涤剂胶束中，或在磷脂脂质体中，但最高百分比的标记发生在无洗涤剂的聚集体。木瓜蛋白酶消化的酶释放的疏水结构域，和聚丙烯酰胺凝胶电泳在十二烷基硫酸钠或凝胶排阻色谱表明，该标签是专门定位在切割的疏水结构域片段。该片段在三步程序中纯化。用附着在Sepharose CL-4 B上的木瓜蛋白酶进行消化，并将上清液吸附到吖啶亲和树脂上以除去亲水性酶片段。然后在Sepharose CL-6 B上层析与Triton X-100胶束缔合的非保留片段，最后在乙醇-甲酸溶剂中通过Sephadex LH-60上层析除去洗涤剂。与肽标准品相比，该片段在Sephadex LH-60柱上表现出3100的表观分子量。然而，纯化片段的氨基酸分析显示，每摩尔片段中组氨酸和甘氨酸各仅为1摩尔，而根据分子量估计预期的氨基酸为25-30摩尔。这一结果表明人红细胞乙酰胆碱酯酶疏水结构域的一种新的非氨基酸结构。乙酰胆碱酯酶（AChE，1 EC 3.1。1.7)在脊椎动物组织中以多种形式存在。如果这些形式含有胶原蛋白样尾，则可将其分类为不对称的，如果它们缺乏这种尾结构，则可将其分类为球形的[参见Massoulié & Bon（1982）和Rosenberry（1985）]。球状乙酰胆碱酯酶包括一类膜结合形式，
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 Received October 25, 1985; Revised Manuscript Received January 14, 1986 abstract: The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(w-[125I] iodophenyl) diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mol of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.. Acetylcholinesterase (AChE, 1 EC 3.1. 1.7) occurs in a va-riety of forms in vertebrate tissues. These forms can be classified as either asymmetric if they contain a collagen-like tail or globular if they lack such a tail structure [see Massoulié & Bon (1982) and Rosenberry (1985)]. The globular AChEs include a class of membrane-bound forms which are the