Incorporation of L-Dopa into C-terminus of a-tubulin in cultured cells and in vivo

24. DENTESANO Y, BISIG C.G , CARBAJAL A, CHESTA ME, AND ARCE C.A.

Huerta Grande

Congreso; XXVIII Congreso anual de la Sociedad Argentina de Investigación en Neurociencias; 2013

SAN

We previously described that L-Dopa can be in vitro incorporated into the C-terminus of α- tubulin by ?tubulin tyrosine ligase? (TTL). After its incorporation, Dopa cannot be released by ?tubulin carboxypeptidase? (TCP), the other enzyme involved in the tyrosination/detyrosination cycle. Dopa-tubulin, was polymerized into microtubules as well as Tyr-tubulin, and both tubulin types disassembled similarly. To monitor and to measure the amount of Dopa incorporated into tubulin we used a method based the analysis of the tyrosination state (% tyrosinated, % detyrosinated tubulin with respect to total tubulin) of samples before and after Dopa incorporation. We now report that Dopa incorporation into tubulin was also demonstrated to occur in cultured cells in the absence of ?de novo? protein synthesis. Furthermore, once incorporated into tubulin of cultured cells, dopa could not be removed by subsequent incubation even in the presence of added tyrosine suggesting that dopa binds irreversibly to the COOH-terminus of α-tubulin blocking posterior incorporation of tyrosine. Similarly, after intracraneal microinjection of L-Dopa in rats and subsequent brain tubulin purification, we found that L-Dopa was incorporated in the COOH-terminal of tubulin.