Conformation and assembly characteristics of tubulin and microtubule protein from bovine brain.
The conformational requirements for the efficient assembly of bovine brain tubulin into microtubules have been investigated by using near-UV circular dichroism. Microtubule protein was prepared by the assembly-disassembly method of Shelanski et al. [Shelanski, M. L., Gaskin, F., & Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 765-768]. Tubulin dimer, isolated from this multiprotein complex by phosphocellulose ion-exchange chromatography in the presence and absence of Mg2+, was compared with tubulin dimer (WT dimer) prepared by the method of Weisenberg & Timasheff [Weisenberg, R. C., & Timasheff, S. N. (1970) Biochemistry 9, 4110-4116]. The tubulin from both dimer preparations showed identical electrophoretic patterns in which high molecular weight protein was undetectable. However, reproducible and significant differences were found in the near-UV CD spectra. Phosphocellulose-treated tubulin resembles the original microtubule protein more closely than does WT dimer, although this latter material has been widely accepted as being representative of the native protein. The phosphocellulose-treated tubulin and WT dimer are not readily interconvertible by simple physical or chemical treatments. The assembly capability of the various tubulin dimer preparations was compared by measuring the enhancement by tubulin dimer of assembly of ring fraction (isolated from microtubule protein by gel filtration of Sepharose 6B). Again phosphocellulose-treated tubulin is found to have more like native microtubule protein than does WT dimer.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D011506:Proteins; D014404:Tubulin