Cool D E, Blum J J
Department of Biochemistry, University of Washington, Seattle 98195.
Mol Cell Biochem. 1993 Nov;127-128:143-9. doi: 10.1007/978-1-4615-2600-1_13.
L. Donovani promastigotes were grown to late-log and 3-day stationary phase to determine the level of protein tyrosine phosphatase activity in crude extracts and in fractions following gel filtration column chromatography. Over 90% of the activity was soluble in a low salt extraction buffer in both phases of growth. Several peaks of activity were resolved following gel filtration of the crude extracts indicating that multiple tyrosine phosphatases are present in these cells. Tyrosine phosphatase activity was lower in 3-day stationary than in late log-phase cells and a reduction in the major peak of activity, eluting in a gel fraction corresponding to an M(r) of approximately 168 kDa, was observed. In vivo tyrosine phosphorylation was revealed by Western blot analysis. The degree of phosphorylation of at least two proteins differed in cells obtained from late log phase cultures as compared with 3-day stationary phase cultures. These observations indicate that changes in the balance between tyrosine phosphorylation and dephosphorylation occur with increasing culture age.
将杜氏利什曼原虫前鞭毛体培养至对数生长后期和3天稳定期，以测定粗提物以及凝胶过滤柱层析后各组分中的蛋白质酪氨酸磷酸酶活性水平。在两个生长阶段，超过90%的活性可溶于低盐提取缓冲液。粗提物经凝胶过滤后出现多个活性峰，表明这些细胞中存在多种酪氨酸磷酸酶。3天稳定期细胞中的酪氨酸磷酸酶活性低于对数生长后期细胞，并且观察到主要活性峰降低，该活性峰在对应于约168 kDa相对分子质量的凝胶组分中洗脱。通过蛋白质印迹分析揭示了体内酪氨酸磷酸化情况。与3天稳定期培养物相比，对数生长后期培养物中获得的细胞中至少两种蛋白质的磷酸化程度有所不同。这些观察结果表明，随着培养时间的增加，酪氨酸磷酸化和去磷酸化之间的平衡会发生变化。
