A monoclonal antibody to alpha-tubulin: purification of functionally active alpha-tubulin isoforms.
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.
Banerjee
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Department of Biochemistry, The University of Texas Health Science Center, San Antonio 78284-7760, USA. ab@bioc02.uthscsa.edu
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002417:Cattle; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D003078:Colchicine / Q000378:metabolism; D005260:Female; D015151:Immunoblotting; D008274:Magnesium / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012685:Sepharose; D017421:Sequence Analysis; D014404:Tubulin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D014404:Tubulin; D012685:Sepharose; D008274:Magnesium; D003078:Colchicine