Keith K, Hide G, Tait A
Wellcome Unit of Molecular Parasitology, University of Glasgow, Scotland, U.K.
Mol Biochem Parasitol. 1990 Nov;43(1):107-16. doi: 10.1016/0166-6851(90)90135-9.
Protein kinase activities in bloodstream and procyclic forms of Trypanosoma brucei have been partially purified and characterised. Cytosolic extracts were separated on DEAE-cellulose and assayed for the ability to phosphorylate histone in the presence of Ca2+ and diacylglycerol. Five peaks of activity were identified in bloodstream T. brucei and only three in procyclic lysates. One of the kinases present in bloodstream T. brucei shares may characteristics with mammalian protein kinase C. Further characterisation of the kinases using an in situ assay after separating proteins by isoelectric focussing confirmed that the kinases present in bloodstream and procyclic stages differed in properties and either bloodstream kinases are more stable or greater in number than procyclic kinases. A protein present in bloodstream T. brucei was shown by Western blot analysis to contain an epitope recognized by a monoclonal antibody raised against mammalian protein kinase C. We thus conclude that the protein kinases are differentially regulated between the two stages of the parasite and that the bloodstream stage has a protein kinase C homologue. The implications of these findings in relation to a cellular signalling pathway in trypanosomes is discussed.
布氏锥虫血流形式和前循环形式中的蛋白激酶活性已得到部分纯化和表征。将胞质提取物在二乙氨基乙基纤维素上进行分离，并在存在钙离子和二酰甘油的情况下测定其磷酸化组蛋白的能力。在布氏锥虫的血流形式中鉴定出五个活性峰，而在前循环裂解物中仅鉴定出三个。布氏锥虫血流形式中存在的一种激酶与哺乳动物蛋白激酶C具有许多共同特征。通过等电聚焦分离蛋白质后，使用原位测定法对激酶进行进一步表征，证实血流阶段和前循环阶段存在的激酶在性质上有所不同，并且血流形式的激酶比前循环形式的激酶更稳定或数量更多。通过蛋白质印迹分析表明，布氏锥虫血流形式中存在的一种蛋白质含有被针对哺乳动物蛋白激酶C产生的单克隆抗体识别的表位。因此，我们得出结论，寄生虫的两个阶段之间的蛋白激酶受到不同的调节，并且血流阶段具有蛋白激酶C同源物。讨论了这些发现对锥虫细胞信号通路的影响。
