Partial purification and properties of frog liver tyrosine aminotransferase.
Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.
Partial purification and properties of frog liver tyrosine aminotransferase. Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.