32S oligomer of tubulin. Resistance to factors suppressing the formation of microtubules

The sensitivity of the 32S-oligomer of tubulin from the brain to factors preventing the formation of microtubules: colchicine, CaCl₂, cooling, and the absence of GTP, was studied. The content of the oligomer in the preparation and the degree of polymerization were estimated by the method of analytical centrifugation. It was shown that at 25°, 10 μM colchicine does not change the content of the oligomer, and 100 μM decreases it only a little. In 1 mM colchicine, the oligomer dissociated, but still incompletely. The polymerization of tubulin was inhibited partially by 10 μM and entirely by 100 μM colchicine. In a concentration of 1 and 10 mM, CaCl₂ did not decompose the oligomer, but even the smaller of these concentrations inhibited polymerization. Lowering the temperature of incubation to 14 or 4°, while entirely suppressing polymerization, had no influence on the content of the oligomer in the preparation. In the tubulin preparations containing a low amount of exogenous GTP (no more than 3x10^-6 M), the usual amount of the oligomer was detected, whereas polymerization (at 25°) requires GTP in a concentration exceeding 10^-4 M. Thus, the reaction of oligomerization of tubulin is relatively resistant to factors preventing assembly of the microtubules. This evidently means that there are at least two types of bonds between tubulin molecules: in the first place, bonds in the microtubules, sensitive to colchicine, Ca²⁺, and cold, and formed only in the presence of nucleoside triphosphates, and in the second place, bonds realized in the 32S-oligomer of tubulin, more stable and not requiring exogenous nucleotides.