Low M G, Huang K S
Rover Physiology Research Laboratories, Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, NY 10032.
Biochem J. 1991 Oct 15;279 ( Pt 2)(Pt 2):483-93. doi: 10.1042/bj2790483.
Mammalian serum and plasma contain high levels of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). Previous studies with crude serum or partially purified GPI-PLD have shown that this enzyme is capable of degrading the GPI anchor of several purified detergent-solubilized cell surface proteins yet is unable to act on GPI-anchored proteins located in intact cells. Treatment of intact ROS17/2.8, WISH or HeLa cells (or membrane fractions prepared from them) with GPI-PLD purified from bovine serum by immunoaffinity chromatography gave no detectable release of alkaline phosphatase into the medium. However, when membranes were treated with GPI-PLD in the presence of 0.1% Nonidet P-40 substantial GPI anchor degradation (as measured by Triton X-114 phase separation) was observed. The mechanism of this stimulatory effect of detergent was further investigated using [3H]myristate-labelled variant surface glycoprotein and human placental alkaline phosphatase reconstituted into phospholipid vesicles. As with the cell membranes the reconstituted substrates exhibited marked resistance to the action of purified GPI-PLD which could be overcome by the inclusion of Nonidet P-40. Similar results were obtained when crude bovine serum was used as the source of GPI-PLD. These data indicate that the resistance of cell membranes to the action of GPI-PLD is not entirely due to the action of serum or membrane-associated inhibitory factors. A more likely explanation is that, in common with many other eukaryotic phospholipases, the action of GPI-PLD is restricted by the physical state of the phospholipid bilayer in which the substrates are embedded. These data may account for the ability of endothelial and blood cells to retain GPI-anchored proteins on their surfaces in spite of the high levels of GPI-PLD present in plasma.
哺乳动物血清和血浆中含有高水平的糖基磷脂酰肌醇特异性磷脂酶D（GPI-PLD）。先前对粗血清或部分纯化的GPI-PLD的研究表明，这种酶能够降解几种纯化的经去污剂溶解的细胞表面蛋白的GPI锚，但无法作用于完整细胞中的GPI锚定蛋白。用免疫亲和层析从牛血清中纯化的GPI-PLD处理完整的ROS17/2.8、WISH或HeLa细胞（或由它们制备的膜组分），未检测到碱性磷酸酶释放到培养基中。然而，当在0.1% Nonidet P-40存在下用GPI-PLD处理膜时，观察到大量的GPI锚降解（通过Triton X-114相分离测量）。使用[3H]肉豆蔻酸标记的变异表面糖蛋白和重组到磷脂囊泡中的人胎盘碱性磷酸酶进一步研究了去污剂这种刺激作用的机制。与细胞膜一样，重组底物对纯化的GPI-PLD的作用表现出明显的抗性，而加入Nonidet P-40可以克服这种抗性。当使用粗牛血清作为GPI-PLD的来源时，也获得了类似的结果。这些数据表明，细胞膜对GPI-PLD作用的抗性并不完全归因于血清或膜相关抑制因子的作用。更可能的解释是，与许多其他真核磷脂酶一样，GPI-PLD的作用受到底物所嵌入的磷脂双层物理状态的限制。这些数据可能解释了尽管血浆中存在高水平的GPI-PLD，但内皮细胞和血细胞仍能在其表面保留GPI锚定蛋白的能力。
