GTP BINDING PROTEINS AND ADENYLYL CYCLASE

ADP核糖化因子(ARF)约为20 kDa鸟嘌呤 核苷酸结合蛋白最初是由它们的能力确定的 增强霍乱毒素催化的体外ADP核糖化 随后显示参与高尔基体中的囊泡运输 和其他细胞隔间。 ARF以GTP结合的形式活跃；结合的GTP水解为GDP， 可能是在GTP水解酶(GTP酶)激活的帮助下 蛋白质(GAP)会导致失活。国内生产总值换取国内生产总值和 其他工人显示，高尔基体能增强再激活能力 在灯盏花素A(BFA)敏感反应中的膜，导致 关于鸟嘌呤核苷酸交换蛋白是BFA目标的提议。 在这些研究中，一种针对ARF的鸟核苷酸交换蛋白(GEP) 从大鼠脾细胞胞浆中高度纯化得到一种布雷菲尔丁A- 不敏感的形式。GEP似乎是一种大约55 kDa的蛋白质 这只占胞浆蛋白的大约0.0006%。 当添加1.5~5 mM的氯化镁时，酶活最大 Mm EDTA)。在稍低浓度的氯化镁下，非催化 核苷酸结合速度较快，未见GEP效应。有了GEP， 80%的ARF1或ARF3在40分钟内与GTP-Gamma结合。结合的速度 占GDP的比例要慢得多。PI、PA或PS(200微米)是必需的 GEP活性在15-20微米时达到一半最大活性。PE、PC、 DMPC/胆酸盐和PIP2无效。GEP刺激释放 只有在未标记的情况下才能从ARF3结合[35S]GTPGammas GTP GammaS、GTP或GDP。替换为GTPGammaS速度更快， 发生在比GDP低的核苷酸浓度。作为GEP 底物，I类ARF比II类或 N-末端甘氨酸的III类和肉豆蔻酰化反应明显 很重要。

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.