Purification and characterization of tubulin-tyrosine ligase from porcine brain.

A tubulin-tyrosine ligase was purified from porcine brains using DEAE-cellulose chromatography, Sepharose-sebacic acid hydrazide-ATP affinity chromatography and Sepharose-tubulin affinity chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 46,000. The apparent molecular weight was 37,000 on a high speed liquid chromatograph equipped with gel filtration columns. The pH optimum for the activity was around 8 and a second peak was observed at around 6.5 8.5 microM ATP or 30 microM tyrosine gave half-maximal activity. The purified enzyme catalyzed the tyrosination of the alpha subunit of tubulin in vitro.