MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEINS

ADP-核糖基化因子构成20-Kda鸟嘌呤家族 核苷酸结合蛋白，其以GTP依赖性方式刺激， 霍乱毒素催化的Gs`的ADP-核糖基化，与 Gs ′和简单胍基化合物以及毒素A1蛋白。 ARFs 在进化上很保守，存在于所有真核生物中， 贾第虫对哺乳动物的作用，并参与细胞内蛋白质运输。 已克隆了六种哺乳动物ARF、两种酵母ARF和两种贾第虫ARF。 所有这些ARF，在E.大肠杆菌，增强型霍乱毒素ADP- 在存在GTP和磷脂/去污剂的情况下进行核糖基转移酶。 ARF具有约65-96%的氨基酸同一性，并含有几个共有氨基酸。 序列的 一般来说，氨基酸序列的差异是 集中在氨基末端区域和羧基的一半附近。 据报道，ARF的氨基端区域对于 膜靶向、与脂质的相互作用和ARF活性。 从HL 60中分离到一个新发现的ARF超家族基因， 以及人类胎儿和大鼠脑cDNA文库，其编码64-Kda的 含18-Kda功能性ARF的鸟嘌呤核苷酸结合蛋白 结构域，称为ARD 1（ARF结构域）。 ARD1 在所有大鼠组织中检测到3.7和4.1 kb的MRNAS， 来源于小鼠、兔和人的组织。 ARD 1高度保守 老鼠和人类之间的区别 ARD 1的ARF结构域包含序列 据信参与鸟嘌呤核苷酸结合和GTP水解 （磷酸盐结合）。 重组ARD 1或ARD 1的ARF结构域， 缺少对应于氨基末端区域的15个氨基酸 ARF以GTP依赖的方式刺激霍乱毒素ADP- 核糖基转移酶活性。 这些结果支持以下结论： ARF蛋白的氨基末端区域不是激活所必需的 霍乱毒素的特性，并且ARF蛋白的特征可能 作为较大哺乳动物蛋白质的结构域出现。

ADP-ribosylation factors constitute a family of 20-Kda guanine nucleotide-binding proteins, which stimulate in a GTP-dependent manner, cholera toxin-catalyzed ADP-ribosylation of Gs`, proteins unrelated to Gs` and simple guanidino compounds as well as the toxin A1 protein. ARFs are evolutionarily well conserved and present in all eukaryotes from Giardia to mammals, and participate in intracellular protein trafficking. Six mammalian ARFs, two yeast ARFs and two Giardia ARFs have been cloned. All of these ARFs, synthesized in E. coli, enhanced cholera toxin ADP- ribosyltransferaes in the presence of GTP and phospholipid/detergent. ARFs share ~65-96% amino acid identity and contain several consensus sequences. In general, differences in amino acid sequence are concentrated near the amino-terminal regions and the carboxyl halves. It was reported that the amino-terminal region of ARF is critical for membrane targeting, interaction with lipid, and ARF activity. A newly recognized gene of the ARF superfamily was isolated from HL60, and human fetal and rat brain CDNA libraries, which encodes a 64-Kda guanine nucleotide-binding protein containing an 18-Kda, functional ARF domain at its carboxyl terminus, termed ARD1 (for ARF domain). ARD1 MRNAS of 3.7 and 4.1 kb were detected in all rat tissues and in MRNAS derived from mouse, rabbit, and human tissues. ARD1 is highly conserved between rat and human. The ARF domain of ARD1 contains sequences believed to be involved in guanine nucleotide-binding and GTP hydrolysis (phosphate binding). Recombinant ARD1 or the ARF domain of ARD1, which lacks the 15 amino acid corresponding to the amino-terminal region of ARFs stimulated, in a GTP-dependent manner, cholera toxin ADP- ribosyltransferase activity. These results support the conclusion that the amino-terminal region of ARF proteins is not required for activation of cholera toxin and that the characteristic features of ARF proteins may occur as domains of larger mammalian proteins.