Roberts W L, Kim B H, Rosenberry T L
Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Proc Natl Acad Sci U S A. 1987 Nov;84(22):7817-21. doi: 10.1073/pnas.84.22.7817.
Acetylcholinesterases (AcChoEases; EC 3.1.1.7) from bovine (Ebo) and human (Ehu) erythrocytes were purified to apparent homogeneity by affinity chromatography. The hydrophobic portion of the glycolipid membrane anchor of each enzyme was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Several cleavage procedures demonstrated that this radiolabel was highly selective for the fatty acid portion of the anchor in both enzymes. The labeled enzymes were digested with phosphatidylinositol (PtdIns)-specific phospholipase C (EC 3.1.4.10), and label release was assessed by polyacrylamide gel electrophoresis. About 85% of the radiolabel was cleaved from Ebo AcChoEase, whereas only 5% was released from Ehu AcChoEase. This finding agrees with a report that Ebo AcChoEase was quantitatively released from intact erythrocytes by PtdIns-specific phospholipase C but Ehu AcChoEase was not [Low, M. G. & Finean, J. B. (1977) FEBS Lett. 82, 143-146]. The two AcChoEases contained comparable amounts of the anchor components ethanolamine, glucosamine, and myo-inositol, but qualitative and quantitative differences were found in the fatty acids. Thin-layer chromatography of radiolabeled fragments generated from Ebo and Ehu AcChoEases by nitrous acid deamination revealed a major difference in the membrane anchors of the two enzymes. The fragment released from Ebo AcChoEase by this procedure comigrated with PtdIns, whereas the corresponding fragment from Ehu AcChoEase had a mobility much greater than that of PtdIns even though it contained myo-inositol and fatty acids. These studies show that 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine is useful for analysis of lipid-containing compounds and indicate that, whereas Ebo AcChoEase contains PtdIns in its glycolipid anchor, Ehu AcChoEase has a different anchor structure, which is resistant to PtdIns-specific phospholipase C. This observation suggests the existence of a class of glycolipid-anchored membrane proteins resistant to this phospholipase.
通过亲和层析法将牛（Ebo）和人（Ehu）红细胞中的乙酰胆碱酯酶（AcChoEases；EC 3.1.1.7）纯化至表观均一。每种酶的糖脂膜锚定的疏水部分用光活化试剂3-（三氟甲基）-3-（间-[125I]碘苯基）二氮杂环丙烷进行放射性标记。几种裂解程序表明，这种放射性标记对两种酶中锚定的脂肪酸部分具有高度选择性。用磷脂酰肌醇（PtdIns）特异性磷脂酶C（EC 3.1.4.10）消化标记的酶，并通过聚丙烯酰胺凝胶电泳评估标记释放。约85%的放射性标记从Ebo乙酰胆碱酯酶上裂解下来，而从Ehu乙酰胆碱酯酶上仅释放出5%。这一发现与一份报告一致，该报告指出PtdIns特异性磷脂酶C可使Ebo乙酰胆碱酯酶从完整红细胞中定量释放，但Ehu乙酰胆碱酯酶则不能[Low, M. G. & Finean, J. B. (1977) FEBS Lett. 82, 143 - 146]。两种乙酰胆碱酯酶所含的锚定成分乙醇胺、葡糖胺和肌醇量相当，但在脂肪酸方面存在定性和定量差异。通过亚硝酸脱氨从Ebo和Ehu乙酰胆碱酯酶产生的放射性标记片段的薄层色谱显示，两种酶的膜锚定存在主要差异。通过该程序从Ebo乙酰胆碱酯酶释放的片段与PtdIns迁移率相同，而来自Ehu乙酰胆碱酯酶的相应片段尽管含有肌醇和脂肪酸，但其迁移率比PtdIns大得多。这些研究表明3-（三氟甲基）-3-（间-[125I]碘苯基）二氮杂环丙烷可用于含脂化合物的分析，并表明，虽然Ebo乙酰胆碱酯酶的糖脂锚定中含有PtdIns，但Ehu乙酰胆碱酯酶具有不同的锚定结构，对PtdIns特异性磷脂酶C具有抗性。这一观察结果表明存在一类对这种磷脂酶具有抗性的糖脂锚定膜蛋白。
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