Journal of Biological Chemistry · 1986 · 261 citations · 35 references
Life SciencesBiochemistryMolecular BiologyChemical KineticsMolecular BiophysicsProtein BiochemistryBinding KineticsEnzyme KineticsLigand Binding KineticsBimolecular Reaction DynamicsBimolecular Reaction KineticsBimolecular Reaction RatesG Protein Subunit ActivationRate LimitingSmall G-protein Signaling
Purified G proteins, whether oligomeric or isolated alpha subunits, exhibit anomalous nucleotide‑binding kinetics. The anomalous kinetics arise because tightly bound GDP remains in the preparations and its dissociation is the rate‑limiting step, and chromatography with 1 M (NH4)2SO4 and 20 % glycerol removes GDP to <0.1 mol per GTPγS site. Removal of GDP restores normal bimolecular kinetics for GTPγS binding.
Purified guanine nucleotide-binding regulatory proteins, as either the oligomers or the isolated nucleotide-binding alpha subunits, display anomalous kinetics of nucleotide binding. This is due to the presence of tightly bound GDP in these preparations. The dissociation of bound GDP is the rate-limiting step for nucleotide binding. GDP can be removed by chromatography in the presence of 1 M (NH4)2SO4 and 20% glycerol, which yields preparations of G proteins that contain less than 0.1 mol of GDP/mol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-binding site. When the GDP is removed, the binding of GTP gamma S displays kinetics consistent with a bimolecular reaction.
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