DiBattiste D, Golubic M, Stacey D, Wolfman A
Department of Vascular Cell Biology and Atherosclerosis, Cleveland Clinic Foundation, Ohio 44195.
Oncogene. 1993 Mar;8(3):637-43.
Neurofibromin, the product of the neurofibromatosis type 1 gene, was found to form a stable complex with immobilized p21c-Ha-ras-GMP-PNP (a non-hydrolyzable GTP analog). This complex, detectable as early as 30 min after addition of crude brain extract, is extremely stable, with less than 50% dissociating after 5 h at 4 degrees C. We interpret this to suggest that the dissociation of full-length neurofibromin from p21c-Ha-ras-GMP-PNP is tightly linked to the hydrolysis of GTP to GDP. Failure to remove a significant proportion of the bound neurofibromin in the presence of EDTA and GDP implies that the binding of neurofibromin to p21c-Ha-ras-GMP-PNP results in the ras protein becoming resistant to guanine nucleotide exchange. Under conditions in which neurofibromin quantitatively binds to p21c-Ha-ras-GMP-PNP, we were unable to detect a complex between p21c-Ha-ras and GAP (GTPase-activating protein). The failure to detect GAP binding to immobilized p21c-Ha-ras-GMP-PNP cannot be explained by the known differences in affinities of the GAP-related domain of neurofibromin and GAP for p21c-Ha-ras-GTP. GAP is, however, able to interact biochemically with immobilized p21c-Ha-ras, suggesting a difference in the interaction between GAP and neurofibromin with p21c-Ha-ras-GMP-PNP.
神经纤维瘤蛋白是1型神经纤维瘤病基因的产物，被发现可与固定化的p21c-Ha-ras-GMP-PNP（一种不可水解的GTP类似物）形成稳定复合物。早在添加粗脑提取物后30分钟即可检测到这种复合物，它极其稳定，在4℃下5小时后解离的不到50%。我们据此推断，全长神经纤维瘤蛋白从p21c-Ha-ras-GMP-PNP上的解离与GTP水解为GDP紧密相关。在存在EDTA和GDP的情况下未能去除相当一部分结合的神经纤维瘤蛋白，这意味着神经纤维瘤蛋白与p21c-Ha-ras-GMP-PNP的结合导致ras蛋白对鸟嘌呤核苷酸交换产生抗性。在神经纤维瘤蛋白定量结合到p21c-Ha-ras-GMP-PNP的条件下，我们无法检测到p21c-Ha-ras与GAP（GTP酶激活蛋白）之间的复合物。未能检测到GAP与固定化的p21c-Ha-ras-GMP-PNP结合，不能用神经纤维瘤蛋白的GAP相关结构域和GAP对p21c-Ha-ras-GTP亲和力的已知差异来解释。然而，GAP能够与固定化的p21c-Ha-ras进行生化相互作用，这表明GAP和神经纤维瘤蛋白与p21c-Ha-ras-GMP-PNP之间的相互作用存在差异。
