Further studies on tubulin polymerization In Vitro

Tubulin was polymerized to microtubules in a medium containing 4 M glycerol-5 mM 2-(N-morpholino)ethanesulfonic acid-0.5 mM MgSO₄-1.0 mM ethyleneglycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid-1.0 mM ATP-50 mM KC1 at pH 6.5 (glycerol reassembly buffer) In Vitro. Partially purified tubulin was obtained by sedimenting these reconstituted microtubules. There were two peaks in the partially purified tubulin fraction upon gel filtration through a Sephadex G-200 column. One was flow-through fraction and the other was purified tubulin dimer (molecular weight 105,000±5,000), having sedimentation coefficients of 20 S and 5.2 S, respectively. One mole of tubulin dimer bound 0.8 mole colchicine and contained α- and β-subunits whose amino acid compositions were determined.Pure tubulin dimer obtained by Sephadex G-200 column chromatography failed to polymerize to microtubules by itself. Addition to the tubulin dimer of a small amount of partially purified tubulin fraction, flow-through fraction or fragmented outer fiber microtubules from sea urchin sperm flagella induced the polymerization of tubulin, as demonstrated by measurements of viscosity changes as well as by electron microscopic observations.