Morita N, Nakazato H, Okuyama H, Kim Y, Thompson G A
Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
Biochim Biophys Acta. 1996 May 21;1290(1):53-62. doi: 10.1016/0304-4165(95)00185-9.
Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [3H]ethanolamine, [3H]myristic acid and [3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.
糖基磷脂酰肌醇（GPI）锚定蛋白广泛存在于动物细胞表面，甚至可能普遍存在，在动物细胞表面发挥着多种重要功能。然而，植物细胞上是否存在GPI锚定蛋白尚未得到证实。本文提供了证据，证明在浮萍少根紫萍中，缺磷会诱导产生一种50 kDa的GPI锚定碱性磷酸酶（AP）。少根紫萍蛋白经Triton X-114分配后产生了两种AP活性形式。与去污剂相关的形式被[3H]乙醇胺、[3H]肉豆蔻酸和[3H]棕榈酸显著标记。这种两亲性形式的AP，与来自哺乳动物的正宗GPI锚定AP一样，通过两种不完全变性的聚丙烯酰胺凝胶电泳，可与其余的水溶性AP活性清晰区分。与溶剂脱脂两亲性AP共价结合的脂质对磷脂酰肌醇特异性磷脂酶C的切割具有抗性。对3H脂肪酸标记的两亲性AP进行强酸或强碱水解，产生放射性脂肪酸和一种暂定为长链碱的放射性脂质。在与[3H]乙醇胺一起孵育的植物中，含量更丰富的水溶性AP也具有放射性，并且被3H脂肪酸标记的程度较低。与它的两亲性对应物不同，水溶性AP通过温和的碱性水解可以去除所有脂肪酸放射性，这表明存在酯连接的脂肪酸。所有证据都支持这样的结论：少根紫萍AP作为一种带有含神经酰胺GPI锚的两亲性蛋白被合成。
