MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEINS

GTP 结合蛋白的分子和生物化学表征

• 批准号: 5203497
• 负责人: J-X HONG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5203497
• 关键词: ADP ribosylation; adenylate cyclase; cholera toxin; conformation; enzyme activity; enzyme structure; genetic library; guanine nucleotide binding protein; guanosine triphosphate; mutant; nucleoside ribosyltransferase; phospholipids; recombinant proteins; structural genes

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.

ADP核糖化因子(ARF)是一个20 kDa的鸟嘌呤核苷酸家族。 结合蛋白被发现是霍乱毒素A的激活剂 亚基(CTA)催化的刺激GTP结合的ADP-核糖化 腺酰环化酶系统的蛋白(Gsα)并参与 细胞内囊泡膜转运。ARF在以下情况下被激活 结合的GDP被GTP取代，并通过结合的GTP的水解而失活 以产生ARF-GDP。通常，ARF被隔离在非活动的GDP绑定中 声明并要求将GTP与洗涤剂或 活性磷脂。缺乏重组ARF1的纯化突变体 前13个氨基酸(rdelta13ARF1-P)刺激霍乱毒素活性 基本上相同，添加或不添加GTP(和磷脂或 洗涤剂)。在本研究中，rdelta13ARF1-P被证明含有结合 核苷酸，后来被鉴定为GTP和GDP。核苷酸- 游离rdelta13ARF1(rdelta13ARF1-F)，7M透析制备 尿素在没有添加GTP的情况下，在没有十二烷基硫酸钠的情况下是活性的，但没有活性 在其存在的情况下不添加GTP。Rdelta13ARF1-F的复性 GTP、ITP、GDP或IDP的存在分别产生， Rdelta13ARF1-GTP和rdelta13ARF1-ITP处于活动状态，以及 Rdelta13ARF1-GDP和rdeltaARF1-IDP，处于非活动状态。这些研究 与氨基末端影响 核苷酸结合和氨基末端截短的ARF可以假定 在没有GTP的情况下的活性构象。