Krust B, Galabru J, Hovanessian A G
J Biol Chem. 1984 Jul 10;259(13):8494-8.
Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous Mr = 67,000 and 72,000 proteins, respectively. Such kinase activity can be assayed after its partial purification on poly(I) X poly(C)-Sepharose. Under these experimental conditions, the apparent km of the kinase for ATP is 1.0 X 10(-6) M and 2.5 X 10(-6) M in enzyme fractions from mouse L-929 and human HeLa cells, respectively. The Mr = 67,000 and 72,000 proteins are phosphorylated by their serine and threonine residues, the ratio of which is modified in preparations from interferon-treated cells. Both of these phosphoproteins are composed of several subspecies with similar isoelectric points (pIs) in the range of 7.2 to 8.2. This heterogeneity is due to the number of phosphate groups per molecule of protein. Accordingly, the pIs of highly phosphorylated proteins are at a less basic pH (7.2 to 7.5). Furthermore, highly phosphorylated proteins show an increase in their apparent molecular weights compared to partially phosphorylated ones. This corresponds to an increase of Mr = 1,500. Partial proteolysis of the 32P-labeled Mr = 67,000 and 72,000 proteins by Staphylococcus aureus V8 protease, alpha-chymotrypsin and thrombin, indicated that these phosphoproteins differ in their polypeptide structure. Phosphorylation of the Mr = 67,000 and 72,000 proteins in enzyme fractions from control L-929 and HeLa cells is enhanced by mixing with extracts from interferon-treated heterologous cells. Proteins, Mr = 67,000 and 72,000, therefore, may serve as suitable substrates for an exogenous kinase, thus indicating that the substrate in enzyme fractions from control cells is less phosphorylated because of a low level of kinase activity.
经干扰素处理的小鼠和人类细胞显示出蛋白激酶活性水平增强，这分别表现为内源性分子量为67,000和72,000的蛋白质发生磷酸化。这种激酶活性在经聚（I）×聚（C）-琼脂糖凝胶部分纯化后即可进行测定。在这些实验条件下，来自小鼠L-929细胞和人类HeLa细胞的酶组分中，该激酶对ATP的表观米氏常数分别为1.0×10⁻⁶ M和2.5×10⁻⁶ M。分子量为67,000和72,000的蛋白质通过其丝氨酸和苏氨酸残基发生磷酸化，在经干扰素处理的细胞制备物中，二者的比例会发生改变。这两种磷酸化蛋白均由几个等电点（pI）相似的亚类组成，范围在7.2至8.2之间。这种异质性是由于每个蛋白质分子的磷酸基团数量不同。因此，高度磷酸化的蛋白质的pI处于碱性较弱的pH值（7.2至7.5）。此外，与部分磷酸化的蛋白质相比，高度磷酸化的蛋白质的表观分子量有所增加。这相当于分子量增加了1,500。用金黄色葡萄球菌V8蛋白酶、α-胰凝乳蛋白酶和凝血酶对³²P标记的分子量为67,000和72,000的蛋白质进行部分蛋白水解，表明这些磷酸化蛋白的多肽结构不同。将来自对照L-929和HeLa细胞的酶组分与经干扰素处理的异源细胞提取物混合后，分子量为67,000和72,000的蛋白质的磷酸化作用增强。因此，分子量为67,000和72,000的蛋白质可能是外源性激酶的合适底物，这表明对照细胞的酶组分中的底物由于激酶活性水平低而磷酸化程度较低。
