CHARACTERIZATION OF THE INVITRO REASSEMBLY OF TUBULIN DERIVED FROM STABLE STRONGYLOCENTROTUS-PURPURATUS OUTER DOUBLET MICROTUBULES

We have characterized the in vitro reassembly properties of tubulin derived from stable outer doublet microtubules of Strongylocentrotus purpuratus sperm tails. These data are of special interest, since previous studies have employed tubulins derived from labile microtubules whose physiological properties are strikingly different from those of stable outer doublets. Reassembly occurred optimally in 5 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM ethylene glycol bis(β-aminoethyl ether)-N, N-tetraacetic acid, 10 mM MgSO4, 150 mM KCl, pH 6.7. In contrast to the physiological growth temperature of 11-15 °C for S. purpuratus, in vitro polymerization of outer doublet tubulin was optimal at 37 °C. The critical tubulin concentration for assembly was 0.55 mg mL-1 at this temperature. Assembly was dependent on GTP (2 mM), and neither GDP nor GMP would substitute. ATP, CTP, and UTP all promoted microtubule formation owing to the presence of a nucleoside diphosphokinase activity. Po-dophyllotoxin poisoned microtubule formation substoichiometrically: only 2-4% of the total number of tubulin dimers were drug-bound when assembly was inhibited by 50%. These data show that the overall similarities between outer doublet and brain tubulin reassembly are remarkable and suggest that the in vitro polymerization properties of tubulins are highly conserved. Since outer doublet tubulin reassembles to give microtubules with very different properties from outer doublet microtubules, it seems unlikely that the intrinsic in vitro polymerization properties of this tubulin determine either microtubule stability or doublet formation. These data suggest that microtubule-associated proteins are of greater importance in determining certain microtubule properties and functions than the tubulin backbone. © 1979, American Chemical Society. All rights reserved.