Localization of the ATP binding site on alpha-tubulin.
The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001921:Brain; D002417:Cattle; D046911:Macromolecular Substances; D010782:Photolysis; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; D014404:Tubulin; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate