GTP BINDING PROTEINS AND ADENYLYL CYCLASE

GTP 结合蛋白和腺苷酸环化酶

• 批准号: 3757603
• 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/3757603
• 关键词: ADP ribosylation; Golgi apparatus; adenosine triphosphate; adenylate cyclase; cell membrane; chemical binding; guanine nucleotide binding protein; hydrolysis; protein purification

ADP-ribosylation factors (ARFs) are about 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 (GDP) was a BFA target. In the studies reported here, a soluble GDP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of [35S]GTPgammaS binding was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GDP had a size of about 700 kDa, the partially purified GDP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became BFA-insensitive, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the BFA target.

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免疫应答的能力来鉴定， 体外霍乱毒素催化的ADP-核糖基化，随后显示， 参与高尔基体和其他细胞中的囊泡运输 隔间 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（GDP）是一种BFA 目标 在这里报道的研究中，可溶性GDP部分地 从牛脑中提纯的 ARF 1和3上的核苷酸交换， 基于毒素测定中ARF活性的增加和 [35 S] GTP γ S结合依赖于磷脂， 磷脂酰丝氨酸比心磷脂更有效。 GEP出现 增加核苷酸交换率，但不影响 ARF对GTP的亲和力。 而粗GDP的规模约为700 部分纯化的GDP在Ultrogel AcA 54上表现为蛋白质 60 kDa。 随着纯化，GEP活性变得对BFA不敏感， 与结论一致，与早期的推论相反， 交换蛋白本身不是BFA靶。