Purification of polymerizable, acetylated and non-acetylated tubulins

CHESTA, ME, CARBAJAL, A, BISIG, CG AND ARCE, CA

Mendoza

Congreso; Sociedad de Investigaciones en Bioquímica y Biología Molecular; 2012

SAIB

Tubulin is acetylated by α-tubulin acetyl transferase on Lys40 of the α-subunit and deacetylated by HDAC6 or Sirtuin-2. Most tubulin purification methods rely on assembly-disassembly cycling of microtubules producing preparations with low (or null) amount of the acetylated isotope. We found that this is due to a deacetylating activity in brain homogenates. HDAC6 is the involved deacetylase since the activity is inhibited by Trichostatin A (TSA) and tubacin but not by nicotinamide. TSA did not influence microtubule polymerization or depolymerization in vitro. We took advantage of these properties of TSA to impede deacetylation of tubulin during the assembly-disassembly steps and to obtain a microtubule preparation enriched in acetylated tubulin. Inhibitory concentration of TSA in the homogenate was 3 µM and 1 µM in subsequent steps. Three-cycles-purified tubulin conserves most of the acetylated tubulin present in the starting supernatant fraction and is free of deacetylating activity. We estimated that about 64% of the tubulin molecules in these preparations are acetylated. We found no differences between both preparations with respect to several tubulin properties and associated proteins. This is the first method described so far that allows large scale purification of polymerizable tubulin containing significant amounts of the acetylated isotype