Moos P J, Fattaey H K, Johnson T C
Center for Basic Cancer Research, Kansas State University, Manhattan 66506-4903, USA.
J Cell Biochem. 1995 Sep;59(1):79-90. doi: 10.1002/jcb.240590110.
Previous attempts to physically separate the cell cycle inhibitory and protease activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non-overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two-fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine-SDS-PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed.
此前，在对纯化的细胞调节唾液糖肽（CeReS）抑制剂制剂进行物理分离细胞周期抑制活性和蛋白酶活性的尝试大多未成功。从DEAE高效液相色谱柱上对抑制剂制剂进行梯度洗脱，可将细胞生长抑制剂与蛋白酶分离，且已证明这两种活性是不同且不重叠的。进一步纯化使CeReS制剂的比生物活性提高了约两倍。从DEAE柱上洗脱下来的主要抑制级分通过三羟甲基氨基甲烷-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（tricine-SDS-PAGE）和微径反相高效液相色谱进行了进一步分析，结果表明其本质上是均一的。通过DEAE高效液相色谱分离出的另外两个级分也没有蛋白酶活性，它们被证明对细胞增殖有抑制作用，很可能代表CeReS抑制剂的修饰变体。对高度纯化的CeReS进行了氨基酸和碳水化合物组成的化学表征，并评估了碳水化合物在细胞增殖抑制、稳定性和蛋白酶抗性中的作用。
