Biochemical determination of tubulin-microtubule equilibrium in cultured cells

✍ Scribed by Richard E. Ostlund Jr.; Joyce T. Leung; Shirley Vaerewyck Hajek

• Book ID: 102984362
• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 897 KB
• Volume: 96
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90568-2

✦ Synopsis

The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by coichicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsu~oxide.

Microtubules were separated by sedimentation at lOO,t@Og for 10 min in a benchtop ultracentrifuge and then depolymerized to tub&n. Colchicine binding to free tubuhn could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent aftinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GHs 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 (iO-6 to 10e5 M) and incubation at 4'C reduced microtubule-derived tubulin to less than 6% of expected.