DROSOPHILA ACETYLCHOLINESTERASE - DEMONSTRATION OF A GLYCOINOSITOL PHOSPHOLIPID ANCHOR AND AN ENDOGENOUS PROTEOLYTIC CLEAVAGE
DROSOPHILA ACETYLCHOLINESTERASE - DEMONSTRATION OF A GLYCOINOSITOL PHOSPHOLIPID ANCHOR AND AN ENDOGENOUS PROTEOLYTIC CLEAVAGE
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DOI：
10.1021/bi00417a038
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发表时间：
1988-08-23
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;MARSHALL, TL;ROSENBERRY, TL
在果蝇乙酰胆碱酯酶（AChE）中存在一个糖肌醇磷脂锚的几个标准。高度纯化的果蝇AChE的化学分析表明，大约一个肌醇残基每个酶亚基。用光活化亲和探针3-（三氟甲基）-3-（m-[125 I]碘苯基）二氮丙啶（[125 I]TID）放射性标记的果蝇AChE检测金黄色葡萄球菌磷脂酰肌醇特异性磷脂酶C（PI-PLC）的选择性切割，该试剂特异性标记糖肌醇磷脂锚定蛋白的脂质部分。消化与PI-PLC释放75%的这种放射性标记的蛋白质。果蝇AChE在十二烷基硫酸钠凝胶电泳显示突出的55-和16-kDa的带和一个微弱的70-kDa的带。[125 I]TID标记定位于55-kDa片段上，表明该片段是蛋白质的C-末端部分。为了支持这一结论，一个敏感的微测序程序，涉及手动Edman降解结合放射甲基化被用来确定残基2-5的16-kDa片段。与果蝇AChE cDNA序列的比较[Hall，L. M. C.的方法，和Spierer，P.（1986）EMBO J. 5，2949-2954]证实了16-kDa片段包括AChE的N-末端。此外，成熟果蝇AChE的N-末端氨基酸的位置与电鳐AChE的N-末端氨基酸的位置非常同源。还证实了在16-和55-kDa片段中存在可辐射甲基化的乙醇胺。因此，果蝇AChE可能包括涉及乙醇胺的第二个翻译后修饰。
The presence of a glycoinositol phospholipid anchor in Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digeston with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [125I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequences [Hall, L. M. C., and Spierer, P. (1986) EMBO J. 5, 2949-2954] confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.