High affinity interactions of GTPgammaS with the heterotrimeric G protein, transducin. Evidence at high and low protein concentrations.
A well known difference in nucleotide binding characteristics between heterotrimeric G proteins and small GTP binding proteins of the Ras superfamily is that the former bind GTP or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) with a much lower affinity (Kd approximately 10(-8)-10(-7) M) than the latter (Kd approximately 10(-11)-10(-10)M). We report here that the alpha subunit of the heterotrimeric G protein transducin (Gt) binds GTPgammaS with an affinity comparable to that of Ras. High affinity binding was suggested by GTPgammaS titrations of rod outer segment samples with Gt concentrations in the range of 7 nM to 300 nM; the results were more consistent with a dissociation constant for GTPgammaS in the subnanomolar range, than with one in the 10(-8)-10(-7) M range typically reported for heterotrimeric G proteins. Equilibrium binding experiments with G protein concentrations in the subnanomolar to nanomolar range confirmed this conclusion and revealed a dissociation constant of 50 pM. Thus, transducin's affinity for GTPgammaS, and by inference, for GTP, appears to be approximately three orders of magnitude higher than previously reported. These results raise the possibility that some results obtained with high concentrations of nucleotide analogues may be due to minute traces of contaminants such as GTP, GTPgammaS, or GTPalphaS, that have high affinities for Gtalpha.
Malinski
J A
JA
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Zera
E M
EM
Angleson
J K
JK
Wensel
T G
TG
D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D016018:Least-Squares Analysis; D011485:Protein Binding; D013462:Sulfur Radioisotopes; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015293:Transducin