Tyrosine Aminotransferase: PURIFICATION AND CHARACTERIZATION

Abstract

Abstract Tyrosine aminotransferase was highly purified from rat livers in which the enzyme had been induced by a glucocorticoid hormone. The preparation eluted from a DEAE-cellulose column as a single peak gave a single precipitin line by the Ouchterlony double diffusion precipitin reaction and sedimented as a single boundary in the ultracentrifuge with an s20,w of 5.9. A molecular weight of 91,000 was determined by equilibrium sedimentation. Despite these indications of homogeneity, three enzymatically active bands were observed in disk-gel electrophoresis. The purified enzyme is yellow in solution and exhibits absorption maxima at 425 mµ and 327 mµ (at slightly acidic pH), which indicate an interaction of the enzyme with its cofactor, pyridoxal phosphate. Addition of tyrosine shifted the 425 mµ peak to 327 mµ, whereas α-ketoglutarate, the keto-acid substrate, reversed this shift. Separation of enzyme from coenzyme could be achieved by dialyzing holoenzyme with tyrosine in 0.1 m phosphate buffer. Addition of pyridoxal phosphate to apoenzyme caused return of the 425 mµ peak and full reconstitution of activity. One mole of pyridoxal phosphate is bound per 94,000 g of enzyme. Tyrosine aminotransferase has a bell-shaped pH-activity curve with a maximum near pH 7.5. The following apparent Michaelis constants were determined: tyrosine, 1.7 x 10-3 m, α-ketoglutarate, 7 x 10-4 m, pyridoxal phosphate, 1.7 x 10-8 m, and pyridoxamine phosphate, 1.5 x 10-7 m. Phosphate was shown to competitively inhibit the binding of the two cofactors to enzyme with Ki values of 1.1 x 10-3 m and 3.4 x 10-3 m for pyridoxal phosphate and pyridoxamine phosphate, respectively.