Purification and characterization of assembly-competent tubulin from Aspergillus nidulans.
We have developed a procedure for purifying assembly-competent tubulin from Aspergillus nidulans. To our knowledge, this is the first report of the purification of assembly-competent tubulin from a filamentous fungus, and the procedure should be of great value in analyzing the large number of alpha- and beta-tubulin mutations that have been isolated and characterized in A. nidulans. Our procedure consists of overproduction of alpha- and beta-tubulin, partial purification by ion-exchange chromatography, and final purification by rounds of assembly and disassembly. We have found that taxol promotes the assembly of A. nidulans tubulin into microtubules, but a higher concentration of taxol is required for maximal assembly of A. nidulans tubulin than is required for brain tubulin. The critical concentration for assembly in the presence of taxol is also significantly higher for A. nidulans tubulin than for brain tubulin. In addition, A. nidulans microtubules that were assembled and maintained in the presence of taxol depolymerized in conditions in which taxol-stabilized mammalian microtubules remain intact. These results suggest that A. nidulans tubulin has a lower affinity for taxol than mammalian tubulin.
Yoon
Y
Y
Department of Molecular Genetics, Ohio State University, Columbus 43210, USA.
Oakley
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001233:Aspergillus nidulans / Q000737:chemistry*; D002417:Cattle; D005656:Fungal Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D008870:Microtubules / Q000737:chemistry*; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism
D005656:Fungal Proteins; D011994:Recombinant Proteins; D014404:Tubulin; D017239:Paclitaxel