Rudoni S, Colombo S, Coccetti P, Martegani E
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Biochim Biophys Acta. 2001 Apr 23;1538(2-3):181-9. doi: 10.1016/s0167-4889(01)00067-2.
The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition 'in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.
CDC25基因产物是酵母中Ras蛋白的鸟嘌呤核苷酸交换因子。最近有人提出，鸟嘌呤核苷酸的细胞内水平可能会影响交换反应。为了验证这一假设，我们测量了不同生长条件下酵母细胞中的核苷酸水平以及Ras2-GTP的相对含量。发现细胞内GTP/GDP比值对生长条件非常敏感：在指数生长期，该比值较高，接近ATP/ADP的比值，但在稳定期开始前迅速下降，在饥饿条件下进一步降低。向葡萄糖饥饿的细胞中添加葡萄糖会导致GTP/GDP比值快速升高。Ras2-GTP的相对含量以平行方式变化，表明其与胞质GTP/GDP比值存在相关性。此外，对纯化的Ras2蛋白进行的“体外”混合核苷酸交换实验表明，GTP和GDP浓度会影响Ras2-GTP负载的程度，这进一步支持了它们可能的调节作用。
