Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods.
Tubulin-tyrosine ligase (TTL), the enzyme responsible for the reversible addition of a tyrosine residue at the carboxyl end of alpha-tubulin, has been purified from porcine brain using a purification scheme based on standard biochemical procedures. The enzyme preparation was nearly homogeneous (purity greater than 95%), was free of tubulin, and could be stored in the presence of glycerol for several months without loss in activity. To develop a more convenient purification of TTL, we have isolated mouse hybridoma cells secreting antibodies to TTL. These monoclonal antibodies recognize TTL not only in brain tissue but also in the liver of various mammals. Monoclonal antibodies isolated from ascites fluid allowed a rapid purification of TTL from a crude brain extract. TTL stayed bound to the immunoaffinity column in 1.5 M NaCl and was eluted with 3 M MgCl2. Highly active TTL was recovered nearly quantitatively at greater than 95% purity and could be stabilized in the presence of glycerol. Glycerol gradient centrifugation, SDS gel electrophoresis and immunoblots identified TTL as a monomeric protein with an apparent polypeptide molecular weight of about 40,000. A one to one complex of TTL with alpha beta-tubulin was observed by gradient centrifugation.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001921:Brain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification; D008870:Microtubules / Q000648:ultrastructure; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D010453:Peptide Synthases / Q000302:isolation & purification*; D013552:Swine
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase