Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources.
Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources.
复制标题
对天然和重组来源的人 GTP 酶激活蛋白进行纯化、表征和蛋白质印迹分析。
DOI：
10.1016/s0021-9258(18)45826-7
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
K. Koths
中科院分区：
文献类型：
--
作者：
R. Halenbeck;W. J. Crosier;R. Clark;F. McCormick;K. Koths
人ras GTP酶激活蛋白(GAP)是一种胞质因子，能刺激正常N-ras p21的GTP酶活性，而对N-ras p21癌基因变异体的GTP酶活性无刺激作用。我们已经从人胎盘组织中纯化了两种形式的天然ras-GAP。除了先前报道的Mr=120,000的I型GAP(1)之外，还回收了等量的具有GAP活性的Mr=95,000的分子，并显示具有II型GAP所需的N-末端序列。胎盘提取物中的这两种GAP形式经分子筛层析分离，似乎具有单体天然结构。利用杆状病毒表达载体在Sf9昆虫细胞内表达重组人I型GAP，分三步纯化10 mg量的GAP。纯化的天然和重组GAP分子的比较表明，在体外GAP实验中，这三个分子都显示了相似的生物学特异性活性。制备了针对纯化的重组GAP的多克隆抗体，并证明其能中和天然GAP和重组GAP的活性。该抗体对免疫印迹法检测天然GAP具有较高的特异性。在人胎盘细胞质提取液中检测到I型和II型GAP的数量大致相同，但当检查其他人体组织时，只观察到I型GAP。
Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP. The two GAP forms in placental extracts were resolved by molecular sieve chromatography and appeared to have a monomeric native structure. Human recombinant type I GAP was produced intracellularly in Sf9 insect cells using a baculovirus expression vector, and 10-mg quantities were purified to homogeneity in three steps. Comparison of the purified native and recombinant GAP molecules revealed that all three displayed similar biological specific activities in an in vitro GAP assay. A polyclonal antibody to purified recombinant GAP was prepared and shown to neutralize the activity of both native and recombinant GAPs. The antibody was also highly specific for the detection of native GAP by Western blot. Type I and II GAP species were detected in approximately equal amounts in cytoplasmic extracts of human placenta, but only type I GAP was observed when other human tissues were examined.