GTP BINDING PROTEINS AND ADENYLYL CYCLASE

GTP 结合蛋白和腺苷酸环化酶

• 批准号: 5203492
• 负责人: S-C TSAI
• 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/5203492
• 关键词: ADP ribosylation; Golgi apparatus; adenosine triphosphate; adenylate cyclase; cell membrane; chemical binding; guanine nucleotide binding protein; guanosinetriphosphatases; hydrolysis; laboratory rat; protein purification; spleen

ADP-ribosylation factors (ARFs) are approximately 20 kDa guanine nucleotide-binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein (GAP) results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A (BFA)-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein was a BFA target. In these studies, a guanine nucleotide-exchange protein (GEP) for ARF has been highly purified from rat spleen cytosol in a brefeldin A- insensitive form. GEP appeared to be an approximateky 55-kDa protein that represents only approximately 0.0006% of the cytosolic protein. The activity was maximal with 1.5 to 5 mM MgCl2 (in the presence of 1 mM EDTA). At slightly lower concentrations of MgCl2, uncatalyzed nucleotide binding was faster and no GEP effect was seen. With GEP, >80% of ARF1 or ARF3 bound GTPgammaS in 40 minutes. The rate of binding of [3H]GDP was much slower. PI, PA, or PS (200 muM) was necessary for GEP activity; half maximal activation was seen with 15-20 muM. PE, PC, DMPC/cholate and PIP2 were ineffective. GEP stimulated the release of bound [35S]GTPgammaS from ARF3 only in the presence of unlabeled GTPgammaS, GTP, or GDP. Replacement by GTPgammaS was faster and occurred at a lower nucleotide concentration than that by GDP. As GEP substrates, Class I ARFs were much better than those of Class II or Class III and myristoylation of the N-terminal glycine was clearly important.

ADP-核糖基化因子（ARF）是约20 kDa的鸟嘌呤 核苷酸结合蛋白最初通过其 增强体外霍乱毒素催化的ADP-核糖基化， 随后显示参与高尔基体中的囊泡运输 和其他细胞区室。 ARF在GTP结合形式下具有活性;结合的GTP水解为GDP， 可能在GTP水解（GTP酶）激活的帮助下， 蛋白（GAP）导致失活。 GDP换GTP， 其他工蜂显示，高尔基体可增强再激活 膜在布雷菲德菌素A（BFA）敏感的反应，导致 鸟嘌呤核苷酸交换蛋白是BFA的靶点。 在这些研究中，ARF的鸟嘌呤核苷酸交换蛋白（GEP） 已从大鼠脾胞质溶胶中高度纯化， 不敏感的形式。 GEP似乎是一个大约55-kDa的蛋白质 其仅代表约0.0006%的胞质蛋白。 活性在1.5至5 mM MgCl 2下最大（在1 mM EDTA）。 在略低浓度的MgCl 2下，未催化 核苷酸结合更快，没有看到GEP效应。 有了GEP， 40分钟内>80%的ARF 1或ARF 3结合GTP γ S。 绑定率 [3 H]GDP增速放缓。 PI、PA或PS（200 μ M）是必需的， GEP活性;在15-20 μ M时观察到半数最大活化。 PE，PC， DMPC/胆酸盐和PIP 2无效。GEP刺激了 仅在存在未标记的 GTP γ S、GTP或GDP。 GTPgammaS替代更快， 发生在较低的核苷酸浓度比GDP。 作为两性平等方案 底物，I类ARF比II类或 III类和豆蔻酰化的N-末端甘氨酸是明确的 重要.