CHARACTERIZATION OF NATIVE AND RECOMBINANT ADP-RIBOSYLATION FACTORS

ADP-核糖基化因子，或ARF，是20 kDa的鸟嘌呤核苷酸结合因子， 这些蛋白质最初是通过增强霍乱的能力而被鉴定出来的， 毒素催化的Gsalpha的ADP-核糖基化，最近已经显示 通过高尔基体参与蛋白质运输。 gtp结合 蛋白质，包括ARF，参与了一个高度调节的循环， 激活和失活。 这是通过绑定GTP来实现的 （活化）和其随后水解成GDP（失活）。 的 这一循环的机制尚不清楚，但对于 了解ARF蛋白的功能。 由于ARF没有 固有的GT3活性，它可能依赖于其他细胞因子， 参与周期。 为了更好地理解这些机制，我们 研究了与特定ARF蛋白结合的核苷酸， 在不同条件下在大肠杆菌中合成， 将这些与蛋白质的活化状态相关联。 之一 ARF蛋白，人ARF 6，当表达为融合蛋白时， 分离的激活状态，这是独立的外源性GTP。 激活是由于存在结合的GTP，其去除导致 恢复GTP依赖的活性。 相反，重组ARF 6以非融合蛋白形式表达，分离出处于失活状态的蛋白， 与约束GDP的关系。 因此，融合蛋白的存在 似乎保护结合GTP的γ-磷酸免于水解 从而纯化活化蛋白。 霍乱毒素ADP-核糖基转移酶活性显示出最佳的 温度明显低于身体核心温度 （约37摄氏度）发现在空肠，其作用部位在 疾病 在磷脂和可溶性脑ARF的存在下， 活性在宽的温度范围内增强;特别地， 在37 ℃下刺激活性。 因此，ARF可以使 生物通过刺激毒素来适应环境条件 在生理温度下的转移酶活性。

ADP-ribosylation factors, or ARFs, are 20 kDa guanine nucleotide-binding proteins that were first identified by their ability to enhance cholera toxin-catalyzed ADP-ribosylation of Gsalpha and have recently been shown to participate in protein trafficking through the Golgi. GTP-binding proteins, including ARFs, participate in a highly regulated cycle of activation and inactivation. This is accomplished by binding GTP (activation) and its subsequent hydrolysis to GDP (inactivation). The mechanisms underlying this cycle are unknown but are important to understanding the function of the ARF proteins. Since ARF possesses no intrinsic GTPase activity, it may rely on other cellular factors for participation in the cycle. To understand better these mechanisms, we have investigated the nucleotides bound to specific ARF proteins that were synthesized in Escherichia coli under different conditions and correlated these with the activation state of the protein. One of the ARF proteins, human ARF 6, when expressed as a fusion protein, was isolated in an activated state which was independent of exogenous GTP. Activation was due to the presence of bound GTP and its removal resulted in restoration of GTP-dependent activity. By contrast, recombinant ARF 6 expressed as a nonfusion protein was isolated in an inactive state due to the presence of bound GDP. Thus, the presence of the fusion protein appeared to protect the gamma-phosphate of bound GTP from hydrolysis resulting in purification of an activated protein. Cholera toxin ADP-ribosyltransferase activity exhibits an optimal temperature significantly below that of body core temperature (approximately 37 degrees C) found in the jejunum, its site of action in disease. In the presence of phospholipids and a soluble brain ARF, activity was enhanced over a wide range of temperatures; in particular, activity was stimulated at 37 degrees C. ARF thus may enable the organism to adapt to environmental conditions by stimulating toxin transferase activity at physiological temperatures.