MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEINS

ADP-核糖基化因子（ARF）是一个20-kDa鸟嘌呤家族， 核苷酸结合蛋白最初是通过其 能够增强霍乱毒素的ADP-核糖基转移酶活性。 ARF是在所有真核细胞中发现的高度保守的蛋白质。 在 至少有六种哺乳动物的ARF基因已被克隆;这些基因可以被归类为 根据蛋白质大小，推导的氨基酸序列， 系统发育分析和基因结构。 来自所有三类的重组ARF激活霍乱毒素催化的 ADP-核糖基化。 在GTPrS存在下的细胞溶质或纯化的ARF， 与磷脂或细胞膜有关。 据报 ARF的氨基末端是其活性的关键区域， 体外和可能体内。 一种肽，其氨基酸序列为 据报道，ARF 1的氨基末端抑制ARF功能，包括 其增强内体融合和高尔基体转运以及 激活霍乱毒素 在这项研究中，重组ARF 1（rARF 1）， rdelta 13 ARF 1（缺少前13个氨基酸的重组ARF 1）和 rPKA 14 ARF 1（rARF 1，其中前14个氨基酸被 cAMP依赖性蛋白激酶催化的前七个氨基酸 亚基）用于评估氨基末端对 ARF增强霍乱胍丁胺ADP-核糖基化的能力 毒素A亚单位 rdelta 13 ARF 1和rdelta 13 ARF 2的GTP依赖性ARF活性 rPKA 14 ARF 1与ARF 1相似，而 霍乱毒素A的半数最大活化作用，rARF 1略高 rdelta 13 ARF 1和rPKA 14 ARF 1的表达水平更高。 这些结果 与ARF 1的氨基末端不是关键的观点一致 作为霍乱毒素的GTP依赖性激活剂。

ADP-ribosylation factors (ARFs) are a family of 20-kDa guanine nucleotide-binding proteins originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin. ARFs are highly conserved proteins found in all eukaryotic cells. At least six mammalian ARFs genes have been cloned; these can be grouped into three classes based on protein size, deduced amino acid sequence, phylogenetic analysis, and gene structure. Recombinant ARFs from all three classes activate cholera toxin-catalyzed ADP-ribosylation. Cytosolic or purified ARFs in the presence of GTPrS, associated with phospholipid or cell membranes. It has been reported that the amino terminus of ARF is a region critical for its activity in vitro and probably in vivo. A peptide with the amino acid sequence of the amino terminus of ARF1 reportedly inhibited ARF function, including its ability to enhance endosome fusion and Golgi transport as well as to activate cholera toxin. In this study, recombinant ARF1 (rARF1), rdelta13ARF1 (recombinant ARF1 lacking the first 13 amino acid) and rPKA14ARF1 (rARF1 in which the first 14 amino acids were replaced by the first seven amino acids of the cAMP-dependent protein kinase catalytic subunit) were used to assess the effect of the amino terminus on the ability of ARF to enhance ADP-ribosylation of agmatine by the cholera toxin A subunit. The GTP-dependent ARF activities of rdelta13ARF1 and rPKA14ARF1 were similar to that ARF1, whereas the GTP requirement for half-maximal activation of cholera toxin A, was somewhat higher for rARF1 than it was for rdelta13ARF1 and rPKA14ARF1. These results are consistent with the view that the amino terminus of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin.