Burlini N, Lamponi S, Radrizzani M, Monti E, Tortora P
Biochim Biophys Acta. 1987 Sep 14;930(2):220-9. doi: 10.1016/0167-4889(87)90034-6.
A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.
通过SDS凝胶电泳测定，已从酵母粗提物中分离出一种65 kDa的磷蛋白。在我们实验室制定的酶纯化过程中，这种磷酸化形式与磷酸烯醇式丙酮酸羧激酶共纯化（托尔托拉，P.，哈诺泽，G.M.和圭里托雷，A.（1985年）《分析生物化学》144，179 - 185）。此外，在Mn2 +存在的情况下，这两种蛋白质都能与5'AMP - 琼脂糖4B紧密结合，而如果用Mg2 +取代Mn2 +，结合则显著降低。这种结合模式与酵母磷酸烯醇式丙酮酸羧激酶众所周知的对Mn2 +的依赖性一致。这些数据表明，65 kDa的蛋白质可能是天然酶的磷酸化产物。此外，尽管这种磷酸化形式不能被抗磷酸烯醇式丙酮酸羧激酶抗体免疫沉淀，但在与抗体预孵育的粗提物中加入蛋白A - 琼脂糖CL - 4B会导致磷酸化形式与树脂结合，从而为其作为天然酶的修饰形式的鉴定提供了免疫学证据。在存在[32P]Pi的情况下生长的细胞提取物中，以及与[γ - 32P]ATP孵育的细胞提取物中，都能检测到相同的65 kDa磷蛋白。此外，用溴化氰或金黄色葡萄球菌V8蛋白酶消化该磷蛋白，分别产生两个和三个片段，这些片段与磷酸烯醇式丙酮酸羧激酶的消化产物相似，再次支持了所提出的鉴定。最后，对65 kDa蛋白质中磷酸化氨基酸的分析表明，磷酸丝氨酸是唯一被标记的磷酸氨基酸。
