CHARACTERIZATION OF NATIVE AND RECOMBINANT ADP-RIBOSYLATION FACTORS

ADP-核糖基化因子（ARF）是20 kDa的鸟嘌呤核苷酸结合因子， 这些蛋白质最初是根据它们增强免疫应答的能力而被鉴定出来的。 霍乱毒素催化的Gs-alpha的ADP-核糖基化。 ARF分离自 各种哺乳动物组织具有不同的磷脂/去污剂， 活性和稳定性的核苷酸要求。 免疫学研究 使用抗ARF多克隆抗体鉴定膜和可溶性 存在于所有哺乳动物组织中的蛋白质;这些蛋白质的相对量 免疫反应蛋白以组织依赖的方式变化。 的事实 六种哺乳动物ARF样蛋白，五种人类和一种牛， 根据它们的cDNA和推测的氨基酸序列进行鉴定， 不同的ARF在不同的组织中占主导地位。 探讨 无论每个ARF对活动的要求是否不同，我们 检查了磷脂/去污剂和GTP浓度对 天然和重组ARF对霍乱毒素的刺激作用。 虽然重组ARF在E. coli能增强ADP- 霍乱毒素的核糖基转移酶活性，最大需要量 两种ARF的ARF活性和GTP结合率不同。 重组 ARF 2和6在所需的GTP浓度方面存在差异， 半最大活性，毒素刺激的时间过程，及其 磷脂/洗涤剂的要求。 总之，这些数据是 与我们的信念一致，即特定ARF的相对差异在于 不同的细胞，个别的ARF可能有不同的功能。

The ADP-ribosylation factors (ARFs) are 20 kDa guanine nucleotide-binding proteins that were first identified based on their ability to enhance the cholera toxin-catalyzed ADP-ribosylation of Gs-alpha. ARFs isolated from various mammalian tissues had different phospholipid/detergent and nucleotide requirements for activity and stability. Immunological studies using anti-ARF polyclonal antibodies identified membrane and soluble proteins present in all mammalian tissues; the relative amounts of these immunoreactive proteins varied in a tissue-dependent fashion. The fact that six mammalian ARF-like proteins, five human and one bovine, have been identified based on their cDNA and deduced amino acid sequences suggested that different ARFs predominate in different tissues. To investigate whether individual ARFs differ in their requirements for activity, we examined the effects of phospholipids/detergents and GTP concentration on the stimulation of cholera toxin by both native and recombinant ARFs. Although recombinant ARFs expressed in E. coli enhanced the ADP- ribosyltransferase activity of cholera toxin, the requirements for maximal ARF activity and GTP binding were different for the two ARFs. Recombinant ARFs 2 and 6 differed in the concentrations of GTP required for half-maximal activity, the time course for toxin stimulation, and their requirements for phospholipid/detergent. In summary,-these data are consistent with our belief that the relative of specific ARFs differ in different cells and that individual ARFs may have different functions.