Reconstitution of microtubules from purified calf brain tubulin

The in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. (1968), Biochemistry 7, 4466-4479; (1970), Biochemistry 9, 4110-4116 as modified by Lee et al. (1973), J. Biol. Chem. 248, 7253-7262, was successful in a medium consisting of 10(-2) M sodium phosphate, 10(-4) M GTP, and concentrations of magnesium ions ranging from 0.5 to 16 X 10(-3) M at 37 degrees. Filaments resembling native microtubules were formed. The filaments are in equilibrium with the associating species of tubulin and the equilibrium can be shifted to depolymerization by lowering the temperature to 20 degrees. Filament formation is inhibited by calcium ions which also cause disassembly of the formed filaments. The effects of calcium ion can be reversed by the addition of ethylenebis-oxyethylenenitrilo)tetraacetic acid. The formation of filaments is favored by the presence of 3.4 M glycerol; only twisted abnormal filaments are observed in the presence of 1 M sucrose. The high molecular weight components observed in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of many tubulin preparations were shown not to be essential for the formation of the filaments.