Toutant J P, Roberts W L, Murray N R, Rosenberry T L
Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106.
Eur J Biochem. 1989 Apr 1;180(3):503-8. doi: 10.1111/j.1432-1033.1989.tb14674.x.
Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.
人红细胞乙酰胆碱酯酶（AChE）两亲性二聚体中的每个催化亚基仅通过糖基肌醇磷脂锚定在质膜中。与其他哺乳动物物种的红细胞AChE不同，人源酶对磷脂酰肌醇特异性磷脂酶C（PtdIns特异性PLC）的直接切割具有抗性。这种抗性是由于肌醇环上存在额外的脂肪酰基链，该链阻断了PtdIns特异性PLC的作用[罗伯茨等人（1988年）《生物化学杂志》263，18766 - 18775]。在本报告中，采用非变性聚丙烯酰胺凝胶电泳来快速明确地区分两亲性AChE（其中每个催化亚基结合一个非离子去污剂胶束）和亲水性AChE（其不与去污剂相互作用）。用羟胺进行碱性处理使人类红细胞AChE脱酰基，使得两亲性AChE对PtdIns特异性PLC敏感，从而释放出亲水性AChE。尽管血清锚定特异性磷脂酶D（PLD）可切割完整的人红细胞AChE锚定物，但根据非变性电泳判断，这种处理并未释放亲水性AChE。在PLD消化之前或之后进行羟胺处理对于实现这种转化是必要的。这些观察结果表明，当人红细胞AChE锚定磷脂中的三个脂肪酰基或烷基中的任何一个保留时，单个去污剂胶束的结合得以维持。对于可在非变性凝胶电泳后鉴定的蛋白质，这些方法既提供了检测对PtdIns特异性PLC有抗性的糖基肌醇磷脂锚定物的方法，也提供了指示这些锚定物中脂肪酰基和/或烷基链的方法。
