Cowles E A, Agrwal N, Anderson R L, Wang J L
Department of Biochemistry, Michigan State University, East Lansing 48824.
J Biol Chem. 1990 Oct 15;265(29):17706-12.
Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7. When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment. The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells.
对纯化的碳水化合物结合蛋白35（CBP35）以及含有CBP35的小鼠细胞提取物进行了二维凝胶电泳分析。对通过在大肠杆菌中表达cDNA克隆获得的重组CBP35进行这样的分析，得到的等电点（pI）值为8.7。当对小鼠3T3细胞提取物进行二维凝胶电泳和免疫印迹时，观察到两个斑点，对应于pI值8.7和8.2。pI 8.2的物种代表CBP35多肽（pI 8.7）通过添加单个磷酸基团进行的翻译后修饰。这一结论基于以下发现：纯化的CBP35含有一种pI 8.2的物种，其被32PO4标记，并且通过碱性磷酸酶处理可转化为未标记的pI 8.7物种。CBP35的磷酸化（pI 8.2）形式在胞质和核组分中均有发现，而未磷酸化（pI 8.7）的物种仅在细胞核中发现。3T3成纤维细胞的静止群体（汇合单层或血清饥饿的稀疏培养物）的特征是磷酸化的CBP35占优势。将相同的细胞刺激进入增殖状态导致磷酸化物种的量增加；然而，更显著的是未磷酸化形式的水平升高，其在静止细胞中几乎检测不到。
