[28] Expression, purification, and measurements of activity of ARNO1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (ARF1)
| Autor: | Sylviane Robineau, Sophie Béraud-Dufour |
|---|---|
| Přispěvatelé: | Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2001 |
| Předmět: |
chemistry.chemical_classification
0303 health sciences [SDV]Life Sciences [q-bio] Membrane lipids 030302 biochemistry & molecular biology Fluorescence assay ADP ribosylation factor 1 Biology medicine.disease_cause 03 medical and health sciences Membrane chemistry Biochemistry medicine [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Nucleotide Lipid vesicle Guanine nucleotide exchange factor Escherichia coli ComputingMilieux_MISCELLANEOUS 030304 developmental biology |
| Zdroj: | Methods Enzymol. Methods Enzymol., 384 (6608), pp.264-271, 2001, ⟨10.1038/384481a0⟩ |
| DOI: | 10.1016/s0076-6879(01)29087-9 |
| Popis: | The expression in Escherichia coli and purification of ARNO1 and its Sec7 domain ARNO1-Sec7 are described in this chapter. Two different kinds of assay can be used to monitor the exchange activity of ARNO1 or ARNO1-Sec7 on ARFI: classical nucleotide binding measurements with radiolabeled nucleotides or a real-time assay based on the large difference between the tryptophan fluorescence of ARF1-GDP and the tryptophan fluorescence of ARF1-GTP. The advantage of the fluorescence assay is its time resolution. However, this assay requires purified proteins for a good signal-to-noise ratio, and cannot detect futile nucleotide exchanges; as such, exchanges are spectroscopically silent. It is suggested that that although both ARFI and ARNO1 are soluble proteins, they interact with membrane lipids. More importantly, these membrane interactions are necessary for the functional interaction between the two proteins. Therefore, membrane lipids (for instance, artificial lipid vesicles) must be included in functional assays. |
| Databáze: | OpenAIRE |
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