Viscometric demonstration of tubulin polymerization

The reconstitution of tubulin to microtubules in vitro has been demonstrated by the measurement of viscosity changes in addition to electron microscopic observations. The viscosity increase in the reassembly buffer was accompanied by a proportional increase in the length of reconstituted microtubules. The half-life time of decay in polymerizability of tubulin was determined at various protein concentrations. The effects of low temperature, colchicine, nucleotides, divalent cations, ionic strength, and the requirement for "nuclei" materials have been investigated. The viscosity dropped sharply to the original level when the temperature was lowered, and 10^-4 M colchicine also induced depolymerization. In a nucleotide-free reassembly buffer, no polymerization could be induced, whereas the viscosity immediately increased on addition of GTP, ATP, ITP, UTP, CTP, TTP, or ADP. Ca²⁺ ions instantaneously caused degradation of reconsituted microtubules, but the effect was completely reversed by adding excess ethyleneglycol-bis(2-aminoethylether)-N, N, N', N'-tetra acetic acid (EGTA). On the other hand, Mg²⁺ ions were an essential requirement for tubulin polymerization. As in actin polymerization, an ionic strength of 0.1 (KCl) favored the reassembly of microtubules. A requirement for "nuclei" for reassembly was also demonstrated by viscosity measurements.