After demonstration of the decarboxylation of 3, 4-dihydroxyphenylalanine(dopa)’to dopamine (I), the pathway for biosynthesis of norepinephrine shown in Fig. 1 was proposed. Although evidence for this pathway was well established by isot, opic procedures (2, 3), it was not until 1960 that Levin, Levenberg, and Kaufman (4) succeeded in isolating and characterizing the third enzyme in the series, dopamine-B-oxidase(@ hydroxylase). The enzyme responsible for converting tyrosine to dopa has, however, proved elusive. The enzyme tyrosinase was considered in this role, but no evidence for it could be obtained in sympathetically innervated tissues. Reports of the enzymatic conversion of tyrosine to catecholamines by tissue slices and minces have generally been unconvincing owing to large and variable blanks with boiled preparations (5, 6). It has now been possible to demonstrate that brain, adrenal medulla, and sympathetically innervated tissues contain a specific hydroxylase that catalyzes the conversion of L-tyrosine to dopa. A short report on the properties of a particle-bound preparation from brain has already appeared (7). The present communication describes the isolation, purification, and charact# erization of a soluble tyrosine hydroxylase from beef adrenal medulla. The soluble tyrosine hydroxylase requires for activity tetrahydropteridine derivatives comparable to those previously observed for phenylalanine hydroxylase (8).

MATERIALS L-Tyrosine-‘4C (uniformly labeled, 250 mc per mmole) a, nd L-tyrosine-3H (398 mc per mmole, generally labeled) were obtained from New England Nuclear Corporation. All samples of radioactive tyrosine were found to contain material that behaved like dopa in its adsorption onto alumina and in its chromatographic properties. This impurity could be removed by adjustment of tyrosine solutions (3 to 5 ml) to pH 8.5, addition of alumina (0.5 g), and stirring for 10 minutes. After the solution was decanted, the treatment with alumina was repeated two more times. After the final treatment with alumina, the solution was adjusted to pH 6.5 and passed through a column, 0.5 X 3 cm, of Amberlite IRC-50 (Na+) buffered to pH 6.5. Tyrosine was not adsorbed under these conditions and appeared in the effluent. The column was washed with 6 ml of water, and the effluent and washing were combined and acidified by adding HCI to a final concentration of 0.1 N. By this procedure,