CHARACTERIZATION OF NATIVE AND RECOMBINANT ADP-RIBOSYLATION FACTORS

天然和重组 ADP-核糖化因子的表征

• 批准号: 3857988
• 负责人: S R PRICE
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3857988
• 关键词: ADP ribosylation; Escherichia coli; G protein; cholera toxin; detergents; enzyme mechanism; genetic manipulation; guanine nucleotide binding protein; guanosine triphosphate; membrane proteins; phospholipids; protein structure function

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.

adp核糖基化因子（ARFs）与20kda的鸟嘌呤核苷酸结合