Mg2+ dependence of guanine nucleotide binding to tubulin.
The relationship between the concentration of Mg2+ and the binding of GDP and GTP to tubulin dimers was investigated by measuring the displacement of the nucleotide bound at the exchangeable site (E-site) by radiolabeled GDP and GTP. A wide range of concentrations of GTP, GDP, and Mg2+ was explored. In the near absence of Mg2+, the affinity of tubulin for GDP was found to be much greater than its affinity for GTP. In the presence of 1.0 mM Mg2+, however, its affinity for GDP was slightly less than for GTP. The results could be quantitatively described in terms of a small number of reversible equilibria. Equilibrium constants, pertaining to measurements at 0 degrees C, in 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.2 mM dithioerythritol, 2 mM EGTA, pH 6.9, were obtained by nonlinear least squares fitting of the data. When the association constant of tubulin for GDP uncomplexed with Mg2+ was taken to be 1.6 X 10(7) M-1, that for uncomplexed GTP was found to be no larger than 1.4 x 10(4) M-1, at least 1100-fold smaller. The association constant of tubulin for the GDP.Mg2+ complex was found to be 2.5-2.7 x 10(7) M-1, while that for the GTP.Mg2+ complex is 6.4-9.0 x 10(7) M-1.
Correia
J J
JJ
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
Baty
L T
LT
Williams
R C
RC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000465:Algorithms; D000476:Alkanesulfonates; D017738:Alkanesulfonic Acids; D000818:Animals; D002417:Cattle; D004533:Egtazic Acid / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D010879:Piperazines; D014404:Tubulin / Q000378:metabolism*
D000476:Alkanesulfonates; D017738:Alkanesulfonic Acids; D006150:Guanine Nucleotides; D010879:Piperazines; D014404:Tubulin; D006153:Guanosine Diphosphate; D004533:Egtazic Acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C008916:piperazine-N,N'-bis(2-ethanesulfonic acid); D008274:Magnesium