Nakamura T, Nakamura K, Stinson R A
Department of Pathology, University of Alberta, Edmonton, Canada.
Arch Biochem Biophys. 1988 Aug 15;265(1):190-6. doi: 10.1016/0003-9861(88)90384-0.
Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], EC 3.1.3.1) expressed in two human osteosarcoma cell lines (Saos-2 and KTOO5) in culture was the tissue nonspecific type and was released from the plasma membrane by phosphatidylinositol (PI) phospholipase C. Despite a difference of 10-fold between the two cell lines in the amount of alkaline phosphatase expressed, the phospholipase solubilized nearly all of the phosphatase from resuspended cells of the two lines. Alkaline phosphatase released with Nonidet-P40 from Saos-2 cells had a Mr of 445,000 by gradient gel electrophoresis in the absence of detergent; that released by PI-phospholipase C was 200,000. The subunit Mr of both solubilized forms was 86,000. Thus, tetrameric alkaline phosphatase in the membrane is attached by a PI-glycan moiety and is converted to dimers when released by PI-phospholipase C. Tunicamycin treatment of Saos-2 cells in culture affected the release of alkaline phosphatase by a high concentration of PI-phospholipase C, but not by a low concentration; both the rate and extent of release were lower from treated cells. However, the enzyme released from the treated cells was in two forms with different molecular weights; it seems that both glycosylated and nonglycosylated dimers were transported to the cell surface and incorporated into the plasma membrane. Glycosylation does not appear to be necessary for alkaline phosphatase to be anchored in the membrane via PI.
在培养的两种人骨肉瘤细胞系（Saos-2和KTOO5）中表达的碱性磷酸酶（正磷酸单酯磷酸水解酶[最适碱性]，EC 3.1.3.1）是组织非特异性类型，可通过磷脂酰肌醇（PI）磷脂酶C从质膜释放。尽管两种细胞系中碱性磷酸酶的表达量相差10倍，但磷脂酶能使两种细胞系重悬细胞中的几乎所有磷酸酶溶解。在无去污剂的情况下，用Nonidet-P40从Saos-2细胞释放的碱性磷酸酶经梯度凝胶电泳测定其Mr为445,000；由PI-磷脂酶C释放的碱性磷酸酶的Mr为200,000。两种溶解形式的亚基Mr均为86,000。因此，膜中的四聚体碱性磷酸酶通过PI-聚糖部分连接，当被PI-磷脂酶C释放时转变为二聚体。用衣霉素处理培养的Saos-2细胞会影响高浓度PI-磷脂酶C对碱性磷酸酶的释放，但不影响低浓度时的释放；处理细胞的释放速率和程度均较低。然而，从处理细胞释放的酶有两种不同分子量的形式；似乎糖基化和非糖基化的二聚体都被转运到细胞表面并整合到质膜中。糖基化对于碱性磷酸酶通过PI锚定在膜中似乎不是必需的。
