Purification and Some Properties of Tyrosine 3‐Monooxygenase from Rat Adrenal

Abstract

Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1600 nmol min-' mg protein-', which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein.Purification of the enzyme caused changes in the kinetic properties of the enzyme. The K,,, for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 pM to 58 pM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V , they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.Tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine : oxygen oxidoreductase (3-hydroxylating)] catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine, which is the initial and rate-limiting step in the biosynthesis of catecholamines such as dopamine, norepinephrine and epinephrine [1,2].

Simple and Accurate Assay for Tyrosine Hydroxylase from Bovine Retina by High-Performance Liquid Chromatography with Fluorescence Detection

“…In order to confirm the reliability of our method, we simultaneously assayed TH in bovine striatum and adrenal medulla and ob tained values (approx. 6 and 10 nmol/min/mg protein, respectively) similar to the results previously published for their specific activi ties [9,10], For the protection of the separa tion column we used a small guard column and could reproduce stable results for at least up to 1,000 assays per guard column. As another advantage of our method, kinetical studies of retinal TH with different concen trations of substrate were easily handled.…”

The Aromatic Amino Acid Hydroxylases

“…All of the eukaryotic enzymes are homotetramers (Nakata and Fujisawa, 1982;Okuno and Fujisawa, 1982;Kappock et al, 1995) with subunit molecular weights of 5 1,000 to 56,000, while bacterial PAH is a monomer of 30,300 (Nakata et al, 1979;Zhao et al, 1994). In the case of PAH, there is an equilibrium between the homodimer and the homotetramer (Kappock et al, 1995), but the other two enzymes have only been described as tetramers.…”

Mass and in situ Molar Activity of Tyrosine Hydroxylase in the Median Eminence

“…(The molecular weight of native TH. a tetramer, is taken as 240,000 daltons [20].) Serum T4 was measured by R1A using Ventrex-coated tubes (Ventrex Laboratories, Portland, Me.).…”