A monoclonal antibody to alpha-tubulin: purification of functionally active alpha-tubulin isoforms.

Abstract

Both alpha- and beta-tubulin exist as numerous isotypic forms that originate from different primary sequences as well as a variety of posttranslational modifications. Recent studies show that tubulin dimers differing in the beta-subunit differ significantly in their subcellular distribution as well as in their functional properties such as assembly, dynamics, conformation, and interaction with antimitotic drugs; however, very little is known about the functional significance of the different alpha-tubulin isoforms and their posttranslational modifications. In an effort to get a better understanding about the alpha-tubulin isoforms, a monoclonal antibody, AYN.6D10, was prepared against the mammalian alpha-tubulin C-terminal sequence Glu-Glu-Gly-Glu-Glu-Tyr. Using an immunoaffinity column, bovine brain tubulin was fractionated into three functionally active alphabeta heterodimers which were identified by immunoblotting with alpha-tubulin-specific antibodies and sequence analysis. Assembly studies in the presence of glycerol and Mg2+ show that one of the fractions, that contains mainly the tyrosinated form of alpha1/2, assembled poorly, while the nontyrosinated form assembled normally. The results indicate that tubulin dimers differing in their alpha-tubulin may differ in their functional properties. Future studies with the isoforms may yield valuable information regarding the role of alpha-tubulin and its posttranslational modifications in regulating microtubule assembly and function in vivo.