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

AGUSTIN CARBAJAL; MARÍA EUGENIA CHESTA; CARLOS GASTÓN BISIG; CARLOS ÁNGEL ARCE

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2013 vol. 449 p. 643 - 648

0264-6021

Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit.Studies of the post-translational acetylation/deacetylation oftubulin using biochemical techniques require tubulin preparationsthat are enriched in AcTubulin (acetylated tubulin) and(for comparison) preparations lacking AcTubulin. Assembly?disassembly cycling of microtubules gives tubulin preparationsthat contain little or no AcTubulin. In the present study wedemonstrated that this result is owing to the presence of highdeacetylating activity in the extracts. This deacetylating activity inrat brain homogenates was inhibited by TSA (Trichostatin A) andtubacin, but not by nicotinamide, indicating that HDAC6 (histonedeacetylase 6) is involved. TSA showed no effect on microtubulepolymerization or depolymerization.We utilized these propertiesof TSA to prevent deacetylation during the assembly?disassemblyprocedure. The effective inhibitory concentration of TSA was3 μM in the homogenate and 1 μM in the subsequent cyclingsteps. By comparison with immunopurified AcTubulin, weestimated that ∼64%of the tubulin molecules in the three cycledpreparations were acetylated. The protein profiles of thesetubulin preparations, as assessed by SDS/PAGE and CoomassieBlue staining, were identical to that of a preparation completelylacking AcTubulin obtained by assembly?disassembly cycles inthe absence of TSA. The tyrosination state and in vitro assembly?disassembly kinetics were the same regardless of the degree ofacetylation.