Guanosine 5'-O-(3-thiotriphosphate) reduces ADP-ribosylation of the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni) by pertussis toxin without causing dissociation of the subunits of Ni. Evidence of existence of heterotrimeric pt+ and pt- conformations of Ni
| Autor: | R D Sekura, Lutz Birnbaumer, Juan Codina, Rafael Mattera |
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| Rok vydání: | 1987 |
| Předmět: |
GTP'
Cations Divalent Macromolecular Substances Protein Conformation Stereochemistry Guanosine GTPase Pertussis toxin Guanosine Diphosphate Biochemistry Divalent chemistry.chemical_compound GTP-Binding Proteins Heterotrimeric G protein Magnesium Pentosyltransferases Virulence Factors Bordetella Molecular Biology Edetic Acid ADP Ribose Transferases chemistry.chemical_classification Adenosine Diphosphate Ribose Guanylyl Imidodiphosphate Guanosine 5'-O-(3-Thiotriphosphate) Cell Biology Thionucleotides Pertussis Toxin chemistry ADP-ribosylation Adenylate Cyclase Toxin Guanosine Triphosphate Adenylyl Cyclases |
| Zdroj: | Journal of Biological Chemistry. 262:11247-11251 |
| ISSN: | 0021-9258 1173-1117 |
| Popis: | The effect of the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the GTP analog which activates the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni), on the pertussis toxin-mediated ADP-ribosylation reaction was studied in detail. Two effects were discerned: a stimulation of the ADP-ribosyltransferase activity of the toxin, akin to what was described for ATP and GDP in a previous report (Mattera, R., Codina, J., Sekura, R., and Birnbaumer, L. (1986) J. Biol. Chem. 261, 11173-11179), and a decrease in the ability of Ni to be a substrate for the activated toxin. Both effects were time-dependent with activation of the toxin being somewhat faster than inactivation of Ni. The effect of the addition of GTP gamma S on Ni was readily reversed by excess GDP and attenuated by increasing EDTA in the medium from 0.35 to 10 mM, suggesting dependence on trace concentrations of a divalent cation. It is suggested that this cation is Mg2+ on the basis that low (5-10 nM) concentrations of Mg2+ are needed for the endogenous GTPase activity of Ni (Sunyer, T., Codina, J., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 15447-15451). Sucrose density gradient analysis of the Ni X GTP gamma S complexes with decreased susceptibility to ADP-ribosylation by pertussis toxin showed the same sedimentation parameters as Ni or Ni X GDP complexes, indicating that the molecule of Ni with GTP gamma S bound is heterotrimetric as opposed to dissociated into alpha i X GTP gamma S plus beta X gamma. Thus, these experiments define two conformations of heterotrimeric Ni: one -pt+, ADP-ribosylated by pertussis toxin, and the other pt-, poorly or not ADP-ribosylated by pertussis toxin. This latter, hitherto unrecognized conformation, is stabilized by the addition of strongly activating guanine nucleotides such as GTP gamma S and guanyl-5'-yl imidodiphosphate and should be important in the train of events that lead from an inactive heterotrimeric Ni to a fully active and dissociated Ni. |
| Databáze: | OpenAIRE |
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