MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEINS

ADP-核糖基化因子（ARF），一个20-kDa鸟嘌呤核苷酸家族， 结合蛋白，被发现作为霍乱毒素A的激活剂 亚基（CTA）催化的ADP-核糖基化的刺激性GTP结合 腺苷酸环化酶系统（Gs α）的蛋白质，并参与 胞内囊泡膜运输。 ARF在以下情况下激活： 结合的GDP被GTP取代，并通过结合的GTP水解而失活 得出ARF-GDP。 通常，ARF是孤立的，在一个不活跃的GDP结合， 说明并要求添加GTP沿着洗涤剂，或 磷脂的活性。 纯化的突变型重组ARF 1缺乏 前13个氨基酸（rdelta 13 ARF 1-P）刺激霍乱毒素活性 基本上等同地，加入或不加入GTP（和磷脂或 洗涤剂）。 在这项研究中，rdelta 13 ARF 1-P显示含有结合的 核苷酸，后来被确定为GTP和GDP。 核苷酸- 游离rdelta 13 ARF 1（rdelta 13 ARF 1-F），通过对7 M透析制备 尿素，在不存在SDS的情况下，在不添加GTP的情况下具有活性，但在不存在SDS的情况下， 而不添加GTP。 rdelta 13 ARF 1-F在大肠杆菌中的复性 GTP、ITP、GDP或IDP的存在分别产生， rdelta 13 ARF 1-GTP和rdelta 13 ARF 1-ITP，它们是活性的， rdelta 13 ARF 1-GDP和rdeltaARF 1-IDP，它们是无活性的。 这些研究 与氨基末端影响 核苷酸结合，氨基末端截短的ARF可以假设 在没有GTP的情况下的活性构象。

ADP-ribosylation factors (ARFs), a family of 20-kDa guanine nucleotide- binding proteins, were discovered as activators of cholera toxin A subunit (CTA)-catalyzed ADP-ribosylation of the stimulatory GTP-binding protein of the adenylyl cyclase system (Gs alpha) and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require the addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (rdelta13ARF1-P) stimulated cholera toxin activity essentially equally, with or without added GTP (and phospholipid or detergent). In this study, rdelta13ARF1-P was shown to contain bound nucleotides, which later were identified as GTP and GDP. Nucleotide- free rdelta13ARF1 (rdelta13ARF1-F), prepared by dialysis against 7M urea, was active without added GTP in the absence of SDS, but inactive without added GTP in its presence. Renaturation of rdelta13ARF1-F in the presence of GTP, ITP, GDP or IDP yielded, respectively, rdelta13ARF1-GTP, and rdelta13ARF1-ITP, which were active, and rdelta13ARF1-GDP and rdeltaARF1-IDP, which were inactive. These studies are consistent with the hypothesis that the amino terminus affects nucleotide binding and that an amino-terminally truncated ARF can assume an active conformation in the absence of GTP.