MODIFICATION OF THE COOH-TERMINAL TYROSINE OF ALPHA-TUBULIN INDUCED by LOW CONCENTRATION OF FREE TYROSINE IN THE CULTURE MEDIUM

PURRO, S.A.; BISIG, C.G.; CONTIN , M.A.; BARRA, H.S. Y ARCE, C.A

Villa Carlos Paz, Cordoba- Argentina

Congreso; Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular (SAIB).; 2001

Alpha-tubulin is biosynthesized with a tyrosine residue at its COOH-terminus that can be removed by tubulin carboxypeptidase and reincorporated by tubulin tyrosine ligase. Majority of microtubules in cells routinely grown in DMEM (wich contain 400 uM tyrosine) are constituted mainly by tyrosinated tubulin (Tyr-tubulin) while detyrosinated tubulin (Glu-tubulin) is scarce. Data where obtained by using immunoblot and immunofluorescence techniques with antibodies specific to Glu-tubulin and Tyr-tubulin. When cells were  cultured in HAM F12K (60 uM tyrosine) during few days, immunoreactivity against Tyr-tubulin decresed practically to zero while total tubulin remained constant and Glu-tubulin did not increase. Tretament of tubulin with carboxipeptidase A produced a significant increase of Glu-tubulin suggesting that a compound was bound to glutamic acid through peptidic linkage at the COOH-terminus. This compound could be a modified tyrosine residue or an amino acid other than tyrosine that tyrosine that the antibody cannot recognize. When cells that had been grown in F12K were supplemented with tyrosine up to 400 uM and growth continued for 24 hs, immunoreactivity to Tyr-tubulin was recovered. Taxol-stabilization of microtubules of cells cultured in F12K led to a significant increment of Glu-tubulin. These result indicate that even when Tyr-tubulin seems to be absent, the liganse and carboxipeptidase are still active. These results were found using several cell lines.