Abstract
Abstract Tyrosine hydroxylase from bovine adrenal medulla has been solubilized and extensively purified. The catalytic properties of the solubilized, purified enzyme are similar to those of the particulate enzyme. The stoichiometry of the reaction catalyzed by the enzyme, as well as its regulatory properties, have been found to vary with different pterin cofactors. The enzyme, for example, is markedly inhibited by its substrate, tyrosine, in the presence of tetrahydrobiopterin, but not in the presence of 6,7-dimethyltetrahydropterin. Purified tyrosine hydroxylase is sensitive to H2O2 that is generated during the nonenzymatic oxidation of tetrahydropterin. The enzyme can be protected by catalase, peroxidase, or Fe2+ from H2O2-mediated inactivation. In the presence of catalase or peroxidase, Fe2+ does not stimulate the reaction. It is concluded that the previous reports that tyrosine hydroxylase is stimulated by Fe2+ can be explained by the known ability of Fe2+ to decompose H2O2
Highlights
We describe the solubilization, partial purification, and some properties of the enzyme from bovine adrenal medulla
6,7-dimcthyltetrahydroptcrin leads to its inactivation as a cofactor in the phenylalanine~hydrosylating system, and that catalase can partially protect against this inactivation. These results indicate that IIZOz generated during the aerobic, nonenzymatic oxidation of tctmhydropterins is capable of the further oxidation of the pterin
There have been several reports describing the partial purification of particle-bound tyrosine hydroxylase from bovine adrenal medulla.[6]
Summary
The standard reaction was carried out in a total volume of 1.0 ml in a 16.mm culture tube at 25” for 15 min without shaking. The reaction mixture contained, in order of addition: water (to make up to volume), 0.500 pmole of L-tyrosine, 100 pmoles of potassium phosphate buffer (final pH = 6.2), 2000 units of catalase, 0.50 pmole of TPNH, sheep liver dihydropteridine reductase (excess), (14 pmoles of 2-mercaptoethanol may be substituted for the dihydropteridine reductase and TPNH (5)), 0.32 pmole of 6,7-dimethyltetrahydropterin, and tyrosine hydroxylase. A fresh solution of the tctrahydropterin in 0.005 M HCl was prepared before each assay. Because of the instability of the tetrahydropterin in neutral solution, the enzyme reaction should be started within 5 min of the addition of the tetrahydropterin
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