Kinetic properties of tyrosine hydroxylase purified from bovine adrenal medulla and bovine caudate nucleus

Abstract

Tyrosine hydroxylase ( l-tyrosine,tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) was highly purified from the soluble fraction of either bovine adrenal medulla or bovine caudate nucleus and the kinetic properties were compared using 6-methyltetrahydropterin or natural (6R)- l -erythro- tetrahydrobiopterin . Two enzyme fractions with different molecular weights from bovine adrenal medulla were separated on a DEAE-Sephacel column; the apparent molecular weights of the first and second enzyme fractions (Fraction I and Fraction II) were estimated to be 280 000 and 390 000, respectively, by Bio-Gel A-1.5 m chromatography. Fraction II was judged to be composed of several enzyme forms with different molecular weights. The molecular weight of the subunit of Fraction I was estimated to be 60 000 by SDS-polacrylamide slab gel electrophoresis. Therefore, the enzyme may be a tetramer. The kinetic properties of the low molecular weight form (Fraction I) and the high molecular weight form (Fraction II) were compared. When 6-methyltetradropterin was used as an artificial cofactor, Fraction II showed two distinct apparent K m values for the pterin cofactor, whereas Fraction I showed a single apparent K m value. Apparent K m values for tyrosine and oxygen were similar in the two enzyme preparations, and neither tyrosine nor oxygen was inhibitory. When (6R)- l -erythro- tetrahydrobiopterin was used as the natural cofactor, Fraction I showed two distinct apparent K m values for tetrahydrohiopterin in the presence of 217 μM oxygen and 50 μM tyrosine, but a single apparent K m value in the presence of 49 μM oxygen and 50 μM tyrosine. Tyrosine at 100 μM and oxygen at 94 μM were inhibitory on Fraction I with tetrahydrobiopterin as cofactor. These results show that two apparent K m values for natural (6R)- l -erythro- tetrahydrobiopterin cofactor can be observed only in air (217 μM oxygen), and that the different enzyme forms have different apparent K m values for the pterin cofactor. Tyrosine hydroxylase activity in bovine caudate nucleus was also separated into two fractions on a DEAE-Sepahcel column; the major peak was purified further and the apparent molecular weight was estimated to be 310 000 on a Bio-Gel A-1.5 m column. When natural (6R)- l-erythro- tetrahydrobiopterin was used as a cofactor, the enzyme showed a single apparent K m value for tetrahydropterin cofactor in the presence of 217 μM oxygen and 20 μM tyrosine. This result shows that tyrosine hydroxylase from caudate nucleus has a similar molecular weight but different kinetic properties compared with the enzyme from the adrenal medulla.