GDP-GTP exchange processes of G{alpha}i1 protein are accelerated/decelerated depending on the type and the concentration of added detergents.
Although detergents have been widely used in G-protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G-proteins. Hence, we analysed the effects of detergents on guanine nucleotide exchange reactions of Galpha(i1). Lubrol PX, a non-ionic detergent, which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPgammaS binding from/to Galpha in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G-proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPgammaS binding, and the binding rate constant was decreased by three orders of magnitude in the presence of 2% cholate. These results demonstrate that the guanine nucleotide exchange reactions of Galpha(i1) are drastically modulated by detergents differently depending on the type and the state (monomeric or micellar) of the detergents and that dissociation of GDP from Galpha(i1) does not necessarily lead to immediate binding of GTP to Galpha(i1) in some cases. These effects of detergents on G-proteins must be taken into account in G-protein experiments.
Kubota
Makoto
M
Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8511, Japan.
Tanaka
Takeshi
T
Kohno
Toshiyuki
T
Wakamatsu
Kaori
K
D020355:Cholates / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects
D020355:Cholates; D003902:Detergents; D044385:GTP-Binding Protein alpha Subunits; D000077423:Polidocanol; D006153:Guanosine Diphosphate; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate