THE MECHANISM OF ACTION OF COLCHICINE Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970 . J . Mol. Biol . 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t112 = 5 .4 min) and incubation with colchicine at 37 °C in the presence of vinblastine sulfate (t112 = 485 min) . The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0 .9 t 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6 .3 X 10 5 liters/mol at 37 °C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (Ki = 1 .3 X 10-e M) . The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin . Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0 .001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity) . The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37 °C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules .