Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol
| Autor: | Martha Vaughan, Su-Chen Tsai, Joel Moss, Ronald Adamik |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
ADP ribosylation factor
GTP' Magnesium Chloride Guanosine Diphosphate Myristic Acid Structure-Activity Relationship chemistry.chemical_compound Cytosol GTP-binding protein regulators GTP-Binding Proteins Animals Guanine Nucleotide Exchange Factors Phospholipids Multidisciplinary ADP-Ribosylation Factors Guanosine 5'-O-(3-Thiotriphosphate) GDP binding Proteins Phosphatidic acid Brefeldin A Rats chemistry Biochemistry Guanosine diphosphate ADP-Ribosylation Factor 1 Myristic Acids Spleen Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 93:305-309 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport. |
| Databáze: | OpenAIRE |
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