Microtubular protein catalytic interactions with nucleotides.
Purified tubulin prepared from pig brain in the absence of added guanine nucleotides contains 1 mol each of GDP and GTP/mol of tubulin. Incubation of a tubulin preparation with inorganic [32P]phosphate results in the incorporation of 32P into tubulin-associated GDP and GTP. Typically, in a 5-h incubation, 0.45 mM 32Pi reacts with 22 muM tubulin to form 3.3 muM [32P]GDP, and 0.3 muM [32P]GTP. The yield of labeled nucleotide is decreased as the result of a hydrolase activity associated with the preparation. The [32P]GTP is exclusively beta-labeled and is therefore formed by phosphorylation of [32P]GDP by a phosphate donor other than inorganic phosphate, most likely by nonradioactive GTP. Added GDP significantly decreases the yield of labeled GTP, but increases the yield of labeled GDP in a manner that is consistent with a partial protection against [32P]GDP hydrolysis, but not consistent with significant additional [32P]GDP formation. Added GMP is an inhibitor of both labeling activities associated with the preparation, although it has no effect on the hydrolase activity. Added ADP (0.4 MM) does not form labeled ADP or ATP and does not influence the labeling of GDP or GTP. The formation of [32P]GDP was shown to occur by an exchange mechanism rather than through net synthesis from GMP and Pi. These results provide evidence for a reversible guanidylation of the protein. The labeling activity is always specifically associated with highly purified tubulin preparations. Microtubular protein preparations are found to catalyze an exchange of oxygen from H218O into inorganic phosphate. Thus, there are two distinct catalytic properties associated with tubulin.
D000818:Animals; D001665:Binding Sites; D001921:Brain; D006023:Glycoproteins; D006150:Guanine Nucleotides; D006160:Guanosine Triphosphate; D008870:Microtubules / Q000032:analysis*; D011485:Protein Binding; D013552:Swine; D014404:Tubulin
D006023:Glycoproteins; D006150:Guanine Nucleotides; D014404:Tubulin; D006160:Guanosine Triphosphate