A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype

CARBAJAL, A.; CHESTA, M.E.; BISIG, C. G.; ARCE, C.A.

PORTLAND PRESS LTD

Lugar: Londres; Año: 2013 vol. 449 p. 643 - 643

ubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly-disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly-disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in