Purification of Growth Inhibitory Material from Bovine Brain

本项目的目的是从生长停滞细胞的细胞表面纯化一种生长抑制物质。将牛脑切碎并悬浮得到脑细胞悬液。用蛋白水解酶或磷脂酶处理可从细胞表面释放生长抑制活性。当脑细胞悬液用磷脂酶C处理时，活性的恢复不如用蛋白酶处理的有效，但重复性更好。然而，由于磷脂酶C价格昂贵，我们选择了蛋白酶进行进一步的实验。用蛋白水解酶处理脑悬液时，酶活性的恢复在不同的重复实验中有所不同，即使进行了广泛的改进试验，也不能解决这个问题。当我们恢复得很好时，就进行了进一步的纯化。同时对测定该酶活性的细胞系进行了检测。最后选择大鼠正常成纤维细胞系和人正常成纤维细胞系。这些细胞是第一次在…中生长然后用生长因子刺激生长，然后添加测试材料。通过将放射性胸腺嘧啶核苷掺入DNA来监测细胞生长抑制情况。从脑细胞表面回收生长抑制活性，用有机溶剂进一步提取，所得沉淀物冷冻干燥。将材料溶解在缓冲液中，然后应用于凝胶过滤柱。在空隙体积(高分子量部分)和保留在柱中的裂解物中都检测到了活性。恢复并浓缩了空隙体积。最后，恢复了吸附在亲和层析柱上的糖的活性。这种最终的材料具有减少对生长停滞的人和大鼠正常成纤维细胞DNA合成的诱导作用。但经聚丙烯酰胺凝胶电泳法和银染法检测，该材料仍含有多种蛋白质。由于所获得的原料量太少，不能进一步纯化，所以应该等待最终的纯化。较少

The purpose of this project is to purify a growth inhibitory material from the cell surface of growth-arrested cells.Bovine brain was minced and suspended to get brain cell suspension. Growth-inhibiting activity was released from the cell surface by treatment with proteolytic enzymes or with phospholipase. Recovery of the activity was found to be less effective but more reproducible when brain cell suspension was treated with phospholipase C than with proteases. However, since phospholipase C was expensive we chose proteases for the further experiments. Recovery of the activity varied among repeated experiment when brain suspension was treated with protease, and this problem could not be solved even after an extensive trial for improvement. When we got good recovery, the further purification was carried out. The cell line used for the assay of the activity was also examined. Finally we chose rat normal fibroblast line and human normal fibroblast line. These cells were first growth arre … More sted and then growth-stimulated with growth factors followed by the addition of the test materials. Growth inhibition was monitored by the incorporation of radioactive thymidine into DNA. Growth inhibitory activity recovered from the brain cell surface was further extracted with organic solvents and the resulted precipitate was lyophilyzed. The material was dissolved in the buffer and applied on gel filtration column. The activity was detected both in the void volume (high molecular weight fraction) and in fractins retained in the column. The void volume was recovered and concentrated. Finally, the activity that adsorbed on the affinity column for a sugar was recovered. This final material had an activity to reduce an induction of DNA synthesis in growth-arrested human and rat normal fibroblasts. However, this material still contained several kinds of proteins as revealed by polyacrylamidgel electrophoresis and silver staining. Since obtained materials were too small amount to purify further, the final purification should be awaited. Less