REGULATION OF GTP BINDING PROTEINS

ARF（ADP-核糖基化因子）蛋白首先通过其 激活霍乱毒素催化的ADP核糖基化的能力。 这些GTP- 结合蛋白，现在已知在细胞内囊泡中起作用， 转运，与GTP结合时有活性，水解时无活性， GDP的 为了激活失活的ARF-GDP，一种鸟嘌呤核苷酸交换蛋白 (GEP)是促进GDP解离和GTP结合所必需的， 它以更高的浓度存在于细胞中。 抑制这种 过程由布雷菲德菌素A（BFA），真菌代谢产物，干扰 几个研究小组已经报道了囊泡转运。 一个700-kDa 含有被BFA抑制的GEP活性的可溶性蛋白复合物 之前沃恩博士的研究小组已经描述过了 部分纯化 产生约60 kDa的BFA不敏感GDP，其增强了ARF 1的结合 和ARF 3（I类）至高尔基体膜。 在纯化第二BFA之后- 不敏感的国内生产总值，可能性仍然存在，无论是东盟地区论坛的国内生产总值是不是 本身是BFA的目标，或者存在BFA不敏感的以及 非洲区域论坛国内生产总值的BFA敏感形式。 尝试分离对BFA敏感的ARF GEP已经取得了成功。 纯化的约700-kDa复合物含有 一种200 kDa的GEP蛋白，被BFA抑制。 就像另一个被净化的 ARF GEP，它显然只在I类中活跃，最好是 豆蔻酰化的ARF蛋白。 与GDP解离一样，GTP水解需要ARF与 另一种蛋白质 ARF GTP酶激活蛋白（GAP）从 大鼠脾胞质溶胶似乎是50-kDa蛋白质的同源二聚体。 的 纯化的ARF GAP的特异性明显比纯化的ARF GAP的特异性宽得多。 ARF GEPs迄今净化，这似乎是专门与类 I ARF（ARF 1和ARF 3）已通过以下方式进行了事后修改： N-末端豆蔻酰化。 差距的光谱 底物甚至可以包括ARL 1，一种未知的GTP结合蛋白， 在氨基酸序列上与ARF 1有53%相同的功能（181 每个人的残基）。 特定磷脂对ARF活性的显著影响 GAP和所有ARF GEP都得到了验证。

ARF (ADP-ribosylation factor) proteins were first identified by their ability to activate cholera toxin-catalyzed ADP-ribosylation. These GTP- binding proteins, now known to function in intracellular vesicular transport, are active with GTP bound, inactive when it is hydrolyzed to GDP. To activate inactive ARF-GDP, a guanine nucleotide-exchange protein (GEP) is required to promote dissociation of GDP and binding of GTP, which is present in cells at higher concentration. Inhibition of this process by brefeldin A (BFA), a fungal metabolite that interferes with vesicular transport has been reported by several groups. A 700-kDa soluble protein complex containing GEP activity that was inhibited by BFA had been described earlier by Dr. Vaughan's group. Partial purification yielded about 60-kDa BFA-insensitive GDP that enhanced binding of ARF1 and ARF3 (Class I) to Golgi membranes. After purifying a second BFA- insensitive GDP, the possibilities remained that either ARF GDP is not itself a target of BFA, or that there exist BFA-insensitive as well as BFA-sensitive forms of ARF GDP. Attempts to isolate a BFA-sensitive ARF GEP have now been successful. A purified about 700-kDa complex contained a 200-kDa GEP protein that was inhibited by BFA. Like the other purified ARF GEPs, it was apparently active only with class I, preferably myristoylated, ARF proteins. Like GDP dissociation, GTP hydrolysis requires ARF interaction with another protein. An ARF GTPase-activating protein (GAP) purified from rat spleen cytosol appeared to be a homodimer of 50-kDa proteins. The specificity of the purified ARF GAP is clearly much broader than that of ARF GEPs thus far purifed, which seem to interact exclusively with class I ARFs (ARF1 and ARF3) that have been post-translationally modified by N-terminal myristoylation. It seems that the spectrum of the GAP substrates could even include ARL1, a GTP-binding protein of unknown function that is 53% identical in amino acid sequence to ARF1 (181 residues in each). Dramatic effects of specific phospholipids on the activities of the ARF GAP and all of the ARF GEPs have been demonstrated.