Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.
Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.
Antonny
B
B
Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Chabre
M
M
D000535:Aluminum / Q000494:pharmacology*; D017607:Aluminum Compounds; D000818:Animals; D001608:Beryllium / Q000494:pharmacology*; D002417:Cattle; D005459:Fluorides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*
D017607:Aluminum Compounds; C035982:beryllium fluoride; D000535:Aluminum; D015293:Transducin; D001608:Beryllium; D005459:Fluorides; C032311:aluminum fluoride