Purification and quantification of acetylated tubulin

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

Huerta Grande (Córdoba)

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biologia Molecular; 2013

Soc. Arg. de Investigacion en Neurociencia

Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies on the role of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations enriched in AcTubulin (acetylated tubulin) and, for comparison, preparations lacking AcTubulin. Assembly?disassembly cycling of microtubules results in tubulin preparations that contain scarce or no AcTubulin. In the present study, we demonstrated that this result is due to the activity of HDAC6 present in the extracts. By inhibiting this HDAC6 activity with Trichostatin A (TSA) during the purification by assembly/disassembly, we obtained a 3 x cycled tubulin preparation that contains about 64% of Ac-tubulin with respect to total. This preparation was shown to have the same protein composition, same tyrosination state and same kinetics of assembly and disasembly as compared with a preparation obtained in the absence of TSA. We also developed a method to estimate the percentage of AcTubulin relative to total tubulin. The method is based on acetylation of a tubulin sample with acetic anhydride, Western blotting stained by anti-AcTubulin antibody, and comparison of the optical density of the AcTubulin band with that of a corresponding sample that was not chemically acetylated.