Enhancement of tubulin assembly as monitored by a rapid filtration assay

The early kinetics of microtubule formation from lamb brain tubulin isolated by affinity chromatography can be followed by a newly developed filter assay. The rapid collection of microtubules on glass fiber filters permits the calculation of the moles of tubulin polymerized. The filter assay gives both a rate and extent of polymerization that are identical to those obtained by turbidity or sedimentation analysis, respectively. The microtubules trapped by the filter are readily depolymerized by cold (t12= 3 min) and slowly by colchicine (t1/2= 32min). Tubulin purified by affinity chromatography requires a high protein concentration (>4 mg/ml) for polymerization. Although 5m glycerol allows polymerization to occur at tubulin concentrations below 2 mg/ml, the maximum amount of microtubule formation is observed at low tubulin concentration when microtubule-associated proteins are present. These proteins are not retained by the affinity resin; however, they can be eluted from diethylaminoethyl-Sephadex by solutions containing 0.3m KCl. Microtubule-associated proteins enhance both the rate of polymerization and the total amount of tubulin polymerized as assessed by the filter assay, suggesting that they are involved in both initiation and elongation of microtubules.