Eccleston J F, Moore K J, Morgan L, Skinner R H, Lowe P N
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, United Kingdom.
J Biol Chem. 1993 Dec 25;268(36):27012-9.
Single turnover and equilibrium binding measurements on the interaction of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPase-activating proteins, p120-GAP and neurofibromin, have been made utilizing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These have enabled the equilibrium dissociation constants (Kd) for their initial binding and the rate constants of the hydrolysis step to be measured. p120-GAP binds to both Ras proteins with a Kd of 17 microM, whereas neurofibromin binds to both Ras proteins with a Kd of 1 microM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydrolysis step of Pro-12 Ras, but the maximal activation at 30 degrees C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, with Gly-12 Ras. The affinity with which p120-GAP and neurofibromin binds to either Gly-12 or Pro-12 Ras protein was decreased dramatically by increasing ionic strength caused by addition of NaCl. The rate constant of the cleavage step of hydrolysis catalyzed by neurofibromin increases with increasing ionic strength, whereas that catalyzed by p120-GAP appears to be unaffected. The high ionic strength within the cell might result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, with p120-GAP being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenicity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.
利用荧光2'(3')O-(N-甲基蒽甲酰基)-核苷酸对Gly-12和Pro-12 Ras.GTP与GTP酶激活蛋白p120-GAP和神经纤维瘤蛋白的催化结构域之间的相互作用进行了单周转和平衡结合测量。这些测量使得能够测定它们初始结合的平衡解离常数（Kd）以及水解步骤的速率常数。p120-GAP以17微摩尔的Kd与两种Ras蛋白结合，而神经纤维瘤蛋白以1微摩尔的Kd与两种Ras蛋白结合。p120-GAP和神经纤维瘤蛋白都增加了Pro-12 Ras的GTP水解步骤的速率常数，但在30℃时的最大激活倍数分别为120倍和560倍，而与Gly-12 Ras相比分别为70000倍和52000倍。添加NaCl导致离子强度增加，显著降低了p120-GAP和神经纤维瘤蛋白与Gly-12或Pro-12 Ras蛋白结合的亲和力。神经纤维瘤蛋白催化的水解切割步骤的速率常数随离子强度增加而增加，而p120-GAP催化的则似乎不受影响。细胞内的高离子强度可能导致总体GTP酶激活蛋白活性比在体外低离子强度条件下测得的活性低得多，其中p120-GAP受到的抑制更严重。Pro-12 Ras的GTP水解速率比正常Ras快2倍。Pro-12 ras的低致癌性由一个模型解释，该模型中水解和交换的内在速率以及GTP酶激活蛋白和交换因子刺激的速率是成纤维细胞中Ras蛋白生物学活性的决定因素。
