STABILIZATION AND PURIFICATION OF TYROSINE AMINOTRANSFERASE FROM RAT-LIVER

CM, carboxymethyl; DEAE, diethylaminoethyl; DTT, dithiothreitol; EDTA, Ethylenediamine tetraacetic acid; HEPPS, Hydroxyethylpiperazinepropanesulfonic acid; PLP, pyridoxal 5’-phosphate; SDS, sodium dodecyl sulfate. Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation be prevented. The enzyme was stabilized by pyridoxal 5-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-glutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65°C) followed by precipitation from 0.20 M sucrose at pH 5.0; and small-scale chromatography on DEAF-cellulose hydroxyapatite and CM-Sephadex C50 at pH 6.0. These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity. © 1990, Taylor & Francis Group, LLC. All rights reserved.