Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro.
Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP. We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100 000 in its native state, and 17 000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism. The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically. Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditions. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification*; D013379:Substrate Specificity; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase