Cain T J, Liu Y, Kobayashi T, Robinson J M
Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, Columbus.
J Histochem Cytochem. 1993 Sep;41(9):1367-72. doi: 10.1177/41.9.8394855.
Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.
碱性磷酸酶（APase）属于一个不断增加的膜相关蛋白家族，该家族通过糖基磷脂酰肌醇（GPI）锚定在脂质双层上。人类中性粒细胞含有与一种新型膜结合区室相关的细胞内APase池。用趋化肽甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）刺激中性粒细胞会导致基本上所有的APase迅速上调至与细胞外介质连续的部位。先用细胞松弛素B（细胞松弛素B）预处理中性粒细胞，然后用fMLP刺激，同样会导致该酶在细胞表面表达，并使细胞形态发生显著改变，但随后质膜的内化作用会降至最低。细胞松弛素B和fMLP预处理已用于中性粒细胞APase的分离和纯化。具体而言，用磷脂酰肌醇特异性磷脂酶C处理中性粒细胞，以从细胞表面释放GPI锚定蛋白。通过在非变性凝胶系统中电泳及随后的电洗脱，从这些制剂的上清液中纯化APase。通过这种方法，我们迅速将中性粒细胞APase纯化至同质；然后将该蛋白用于免疫。免疫印迹、酶联免疫吸附测定和免疫细胞化学定位用于鉴定所得抗体。
