Hide G, Graham T, Buchanan N, Tait A, Keith K
Department of Veterinary Parasitology, Glasgow University Veterinary School.
Parasitology. 1994 Feb;108 ( Pt 2):161-6. doi: 10.1017/s0031182000068256.
Autophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in gamma 32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cycle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.
蛋白激酶的自磷酸化被认为是哺乳动物细胞信号转导和生长调节途径中的一种重要控制机制。我们着手研究在布氏锥虫中是否能发现任何此类自磷酸化蛋白激酶活性。为了做到这一点，我们开发了一种利用体外测定法来表征此类蛋白激酶活性的系统。该测定通过使用等电聚焦凝胶电泳对锥虫裂解物进行分级分离，随后将凝胶在γ-32P标记的核苷酸三磷酸中孵育并进行放射自显影来进行。我们鉴定出了两类自磷酸化蛋白激酶活性。在第一类中，所有激酶都依赖ATP作为磷酸供体底物，并且都被发现分子大小为60 kDa。在血流期和前循环生命周期阶段之间观察到了这些蛋白激酶活性的差异。此外，向血流期裂解物中添加哺乳动物表皮生长因子会刺激额外的活性。第二类自磷酸化蛋白激酶利用GTP作为磷酸供体，并且都被发现大小为90 kDa。在这些蛋白激酶的活性中也观察到了阶段特异性差异。
