Synthesis of azidotubulin: a photoaffinity label for tubulin-binding proteins.
A photoaffinity label for the identification of tubulin-binding proteins was synthesized from phosphocellulose-purified bovine brain tubulin and (N-hydroxysuccinimidyl)-4-azidosalicylic acid. The azidotubulin derivative retained the ability to undergo temperature-dependent microtubule assembly and disassembly. When incubated with purified tau protein, the azidotubulin and tau formed cross-linked complexes upon photoactivation. When 125I-labeled azidotubulin was used to photoaffinity label tubulin-binding proteins within the kinetochore of isolated mammalian chromosomes, a 130-kDa band was identified on autoradiographs of SDS-polyacrylamide gels of the 125I-labeled azidotubulin/chromosome preparations. The 130-kDa complex was isolated by antitubulin affinity chromatography and analyzed by immunoblotting using both antitubulin and kinetochore-specific sera obtained from human patients with the autoimmune disease scleroderma CREST. The immunoblots demonstrated that the 130-kDa band that was observed on autoradiographs was a complex of a subunit of the tubulin dimer and an 80-kDa CREST-specific kinetochore protein. The binding of azidotubulin to the 80-kDa kinetochore protein was significantly decreased when chromosomes were treated with a mixture of 9 parts underivatized tubulin to 1 part azidotubulin prior to photolysis. The formation of the 130-kDa azidotubulin/kinetochore protein complex was not inhibited by pretreating the chromosomes with CREST serum prior to incubation with azidotubulin. Azidotubulin should be a useful probe for the identification and characterization of tubulin-binding proteins.
Balczon
R D
RD
Department of Cell Biology and Anatomy, University of Alabama, Birmingham 35294.
Brinkley
B R
BR
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D014404:Tubulin