Biochemical determination of tubulin-microtubule equilibrium in cultured cells

Abstract

The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH 3 cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34–41% of tissue culture cell tubulin. Colchicine (10 −6 to 10 −5 m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.