Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.16.2 (tyrosine hydroxylase)
 14,760 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chronic nutritional iron deficiency of 2 to 5 weeks duration reduced the blood hemoglobin content to 30-50% of control values and resulted in an increase in rat adrenal tyrosine hydroxylase (TH) (EC 1.14.16.2) activity. Kinetic and mixing experiments indicated that this increase was due to an increase in enzyme protein. The body weight of iron-deficient rats ranged from 60 to 80% of control; this factor, however, was not responsible for the increase in adrenal TH as enzyme activity was directly proportional to final body weight. To determine whether the increase in adrenal TH in iron-deficient rats was due to increased sympathetic activity to the adrenal medulla, the splanchnic nerve was cut. The increased TH was still observed after adrenal denervation; this indicates that the mechanism of response to iron deficiency lies within the adrenal itself. Age of the rats is important in determining whether the increase in TH activity will occur.
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PMID:The effect of chronic iron deficiency on adrenal tyrosine hydroxylase activity. 1 18

Bovine adrenal tyrosine hydroxylase has been obtained in a form that is 85 to 90% pure. Sodium dodecyl sulfate-gel electrophoresis and density gradient centrifugation studies have established that the subunit molecular weight of the chymotrypsin-solubilized enzyme is 34,000. The presence of iron in the purified enzyme (0.50 to 0.75 mol of iron/mol of enzyme) has been established. Crude particulate tyrosine hydroxylase can be activated by the phospholipid, phosphatidyl-L-serine, or by exposure to enzymatic phosphorylating conditions. Both forms of activation lower the Km of the enzyme for its 2-amino-4-hydroxypteridine cofactor. By contrast, tyrosine hydroxylase that has been solubilized by chymotrypsin cannot be activated by either of these methods.
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PMID:Bovine adrenal tyrosine hydroxylase: purification and properties. 1 85

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.
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PMID:Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications. 135 68

Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5. Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum. Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit). The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex. In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit. In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase. The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.
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PMID:Regulation of recombinant rat tyrosine hydroxylase by dopamine. 135 65

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.
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PMID:The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy. 135 66

Iron, a transition metal possibly involved in the pathogenesis of Parkinson's disease, was tested for its toxic effects toward cultures of dissociated rat mesencephalic cells. When cultures were switched for 24 h to serum-free conditions, the effective concentrations of ferrous iron (Fe2+) producing a loss of 50% of dopaminergic neurons, as quantified by tyrosine hydroxylase (TH) immunocytochemistry, TH mRNA in situ hybridization, and measurement of TH activity, were on the order of 200 microM. High-affinity dopamine (DA) uptake, which reflects integrity and function of dopaminergic nerve terminals, was impaired at significantly lower concentrations (EC50 = 67 microM). Toxic effects were not restricted to dopaminergic neurons inasmuch as trypan blue dye exclusion index and gamma-aminobutyric acid uptake, two parameters used to assess survival of other types of cells present in these cultures, were also affected. Protection against iron cytotoxicity was afforded by desferrioxamine and apotransferrin, two ferric iron-chelating agents. Normal supplementation of the culture medium by serum proteins during treatment was also effective, presumably via nonspecific sequestration. Potential interactions with DA were also investigated. Fe2+ at subtoxic concentrations and desferrioxamine in the absence of exogenous iron added to the cultures failed to potentiate or reduce DA cytotoxicity for mesencephalic cells, respectively. Transferrin, the glycoprotein responsible for intracellular delivery of iron, was ineffective in initiating selective cytotoxic effects toward dopaminergic neurons preloaded with DA. Altogether, these results suggest (a) that ferrous iron is a potent neurotoxin for dopaminergic neurons as well as for other cell types in dissociated mesencephalic cultures, acting likely via autoxidation into its ferric form, and (b) that the presence of intra- and extracellular DA is not required for the observed toxic effects.
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PMID:Toxic effects of iron for cultured mesencephalic dopaminergic neurons derived from rat embryonic brains. 161 93

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.
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PMID:Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions. 167 67

Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5.
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PMID:Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme. 196 6

Bovine adrenal tyrosine hydroxylase (TH) is isolated in a partially inhibited state with the feed-back inhibitors adrenaline and noradrenaline tightly coordinated to high-spin (S = 5/2) Fe(III) at the active site. In addition to the charge-transfer interaction with iron, an additional charged group in the polypeptide chain, with an apparent pKa of about 5.3 at 4 degrees C, is involved in the binding of catecholamines. Protonation of this group increases the pseudo-first order rate constant for the dissociation of the TH-[3H]noradrenaline complex more than 100-fold at 4 degrees C. At pH 7.0 and 30 degrees C, phosphorylation of Ser-40 causes a 6-fold increase in the rate constant for this dissociation.
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PMID:pH-dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation of Ser-40. 197 Jul 88

The role of iron in schizophrenia (SC) has aroused attention because of its modulatory effect on the dopamine receptor and its role as a cofactor for tyrosine hydroxylase. In addition, several postmortem studies suggest that increased mineralization (especially iron) of the basal ganglia is a possible clinicopathological correlate of schizophrenia. In order to quantitate the in vivo mineral content in the basal ganglia of patients with SC, a protocol was developed to analyze CT scans films with a LOATS computer analysis system. A total of 725 consecutive CT scans (275 SC, 450 nonSC) from a psychiatric population were reviewed. Eighteen scans (2.3%) revealed basal ganglia mineralization of which 7 cases carried a diagnosis of SC and 11 had other psychiatric disorders. All subjects had received neuroleptics, and 8 of the 11 patients in the nonschizophrenic group were demented. Both the SC and nonSC patients exhibited a prevalence (2.5%) of basal ganglia mineralization similar to that found in a postmortem series of the general population.
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PMID:No difference in basal ganglia mineralization between schizophrenic and nonschizophrenic patients: a quantitative computerized tomographic study. 201 34


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