Klebe C, Bischoff F R, Ponstingl H, Wittinghofer A
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Biochemistry. 1995 Jan 17;34(2):639-47. doi: 10.1021/bi00002a031.
The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of the rate of p21ras and is even lower in the Ran(Q69L) mutant. Under saturating conditions the rate constant for the RanGAP1 stimulated GTPase reaction is 2.1 s-1 at 25 degrees C, which is a 10(5)-fold stimulation, whereas RanGAP1 has no effect on Ran(Q69L). The intrinsic guanine nucleotide dissociation rates of Ran are also very low and are likewise increased 10(5)-fold by the exchange factor RCC1. Methods to describe the reaction kinetically are presented. The Ran(T24N) mutant, which is analogous to the S17N mutant of p21ras, has decreased relative affinities for both GDP/GTP and favors GDP binding. However, it was found to interact almost normally with RCC1. The combination of these properties leads to stabilization of the Ran(T24N)-RCC1 complex and may result in vivo in depletion of RCC1 available for stimulating guanine nucleotide exchange.
Ran是一种与Ras相关的核蛋白，利用不同的荧光技术研究了其鸟嘌呤核苷酸解离和GTP酶反应，以确定这些反应是如何被鸟嘌呤核苷酸交换因子RCC1和另一种调节蛋白RanGAP1（GTP酶激活蛋白）刺激的。Ran的内在GTP酶活性是p21ras的十分之一，在Ran（Q69L）突变体中甚至更低。在饱和条件下，RanGAP1刺激的GTP酶反应在25℃时的速率常数为2.1 s-1，这是10^5倍的刺激，而RanGAP1对Ran（Q69L）没有影响。Ran的内在鸟嘌呤核苷酸解离速率也非常低，同样被交换因子RCC1提高了10^5倍。文中介绍了动力学描述反应的方法。与p21ras的S17N突变体类似的Ran（T24N）突变体对GDP/GTP的相对亲和力降低，且更倾向于结合GDP。然而，发现它与RCC1的相互作用几乎正常。这些特性的组合导致Ran（T24N）-RCC1复合物的稳定，并可能在体内导致可用于刺激鸟嘌呤核苷酸交换的RCC1耗尽。
