CHARACTERIZATION OF NATIVE AND RECOMBINANT ADP-RIBOSYLATION FACTORS

ADP核糖化因子，或ARF，是20 kDa鸟嘌呤核苷酸结合的 最初通过增强霍乱的能力来鉴定的蛋白质 毒素催化的Gsalpha的ADP-核糖化反应以及最近的研究 通过高尔基山脉参与蛋白质交易。GTP绑定 包括ARF在内的蛋白质参与了一个高度受调控的循环 激活和失活。这是通过绑定GTP实现的 (活化)以及随后的国内生产总值(GDP)(失活)。这个 这个循环背后的机制是未知的，但对 了解ARF蛋白的功能。由于ARF没有 固有的GTP酶活性，它可能依赖于其他细胞因素 在周期中的参与。为了更好地了解这些机制，我们 已经研究了与特定的ARF蛋白结合的核苷酸 在不同条件下在大肠杆菌中合成， 将这些与蛋白质的激活状态相关联。其中一个 ARF蛋白，人ARF 6，当作为融合蛋白表达时， 在不依赖于外源GTP的激活状态下分离。 活化是由于结合的GTP的存在，并导致其去除 恢复GTP依赖的活性。相比之下，重组ARF 6以非融合蛋白的形式表达，处于非活性状态 到绑定的GDP的存在。因此，融合蛋白的存在 似乎保护了结合的GTP的伽马磷酸盐不被水解 从而得到一种活性蛋白质的纯化。 霍乱毒素ADP-核糖基转移酶活性最高 体温明显低于身体核心温度 (约37摄氏度)在空肠中发现，其作用部位 疾病。在磷脂和可溶性脑ARF存在的情况下， 在广泛的温度范围内，活性得到了增强；尤其是， 活动在37摄氏度被刺激。因此，ARF可以使 有机体通过刺激毒素来适应环境条件 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.