Microtubule formation from two components separated by gel filtration of a tubulin preparation.
(1) A tubulin preparation, purified by two cycles of polymerisation in 4 M glycerol, was further fractionated into two components by chromatography on a column of 6% agarose. One was a fraction of pure tubulin dimer devoid of any combination of high molecular weight ingredients (component T). The other was an aggregate of tubulin containing several minor ingredients (component N). (2) Microtubule formation from these two components was followed in a quantitative way by measuring flow birefringence (deltan). When component N was incubated at 37 degrees C, an instantaneous increase of delta n was observed even at a low concentration of protein, and the extent of polymerisation was roughly proportional to the protein concentration. With component T, the polymerisation occurred after a lag period, and only at a protein concentration higher than at least 0.5 mg/ml. Polymerisation of component T was greatly accelerated when a small amount of component N was added to the reaction medium. (3) Component N was dissociated into a tubulin dimer when the ionic strength of the medium was increased. On reducing the ionic strength, the dimer was reassociated to form the aggregate, which was again capable of accelerating polymerisation of component T. Minor ingredients contained in the component N were not completely removed during the course of its dissociation and reassociation. The dynein-like protein that was present in the component N, however, was no longer detectable in the reassociated aggregate.
D000818:Animals; D000838:Anions; D001665:Binding Sites; D001718:Birefringence; D001923:Brain Chemistry; D002122:Calcium Chloride; D002850:Chromatography, Gel; D003078:Colchicine; D004492:Edetic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D008623:Mercaptoethanol; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D009419:Nerve Tissue Proteins; D009994:Osmolar Concentration; D011189:Potassium Chloride; D011485:Protein Binding; D013552:Swine; D013997:Time Factors
D000838:Anions; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D008623:Mercaptoethanol; D011189:Potassium Chloride; D004492:Edetic Acid; D002122:Calcium Chloride; D003078:Colchicine