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)

This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 X 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 micrograms protein with 150 microM L-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37 degrees C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 micrograms hog kidney aromatic-L-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37 degrees C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37 degrees C may reach 14,000 dpm, whereas the blank only has 300 dpm (less than 3% of test value). This microassay is fast (less than 2 h to complete all reactions) and convenient for performing a large number of determinations.
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PMID:A simplified 14CO2-trapping microassay for tyrosine hydroxylase activity. 287 71

The innervation of human axillary sweat glands was studied by using the specific SPG (sucrose-potassium phosphate-glyoxylic acid) catecholamine histofluorescence method and the peroxidase-antiperoxidase (PAP) immunocytochemical method. The present results demonstrated that human sweat glands are surrounded by nerves containing a weak tyrosine hydroxylase activity. Nerves showing catecholamine histofluorescence could be visualized around the sweat glands only in the presence of exogenous catecholamine (adrenaline in the local anestheticum). In all tissue specimens studied fluorescent adrenergic nerves could be seen around arteries and arterioles corresponding to the distribution of neuropeptide Y-like immunoreactive nerves.
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PMID:The distribution of sympathetic adrenergic, tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerves in human axillary sweat glands. 287 46

The effects of inositol phospholipids on adrenal tyrosine hydroxylase (TH) were studied. Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) caused a rapid and concentration-dependent activation of TH in vitro. The potency of this activation was dependent on the number of phosphate groups in the lipid molecule, and the activation was completely suppressed by increasing the concentrations of salts in the reaction mixture. These results seem to indicate that the activation of TH by inositol phospholipids may be due to their electrostatic action, and suggest a possible involvement of inositol phospholipids in the regulation of TH activity in vivo.
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PMID:Inositol phospholipids cause the activation of adrenal tyrosine hydroxylase through their electrostatic action on the enzyme. 287 6

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.
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PMID:Phosphorylation of tyrosine hydroxylase by cyclic GMP-dependent protein kinase. 287 92

A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) (L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit Mr of 60,000 and a specific activity of 425 nmol.min-1.mg-1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 +/- 0.09 mol iron, 0.13 mol zinc and 0.62 +/- 0.04 mol phosphate per mol subunit of Mr = 60,000. A broad light absorption band with its maximum around 700 nm (epsilon 700 nm = 1.3 (mM monomer)-1.cm-1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) (S = 5/2) in an environment of nearly axial symmetry (g values at 7.2-6.7, 4.7-5.3 and 1.9-2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.
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PMID:Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties. 289 60

Effects of the 1-methyl-4-phenylpyridinium ion (MPP+) on DOPA formation and phosphorylation of tyrosine hydroxylase (TH) of rat pheochromocytoma PC12h cells were examined after the cells were cultured with MPP+. DOPA formed from endogenous tyrosine in PC12h cells after a 3-day culture with 100 microM MPP+ was decreased to less than 50% as compared to that in the control cells cultured without MPP+. Kinetical study showed that two apparent forms of TH with different Km existed in the cells cultured with 100 microM MPP+ but one form in that of control. Incorporation of radioactive phosphate into TH molecule was also reduced to 50% of its control value following a 3-day exposure to 100 microM MPP+. These results suggest that MPP+ acutely inhibits the phosphorylation of TH to decrease cellular DOPA formation.
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PMID:Effect of the 1-methyl-4-phenylpyridinium ion on phosphorylation of tyrosine hydroxylase in rat pheochromocytoma PC12h cells. 289 10

The formation of vertebrate neural circuitry is regulated in part by neurotrophic agents, such as nerve growth factor (NGF); however, the biochemical mechanisms involved in neurite outgrowth have yet to be completely resolved. Phorbol ester tumor promoters are known to influence the extension of neurites in a variety of neurodevelopmental systems, and protein kinase C, the major phorbol ester receptor, has been implicated in this process. In the present study, sphingosine, a specific pharmacological inhibitor of protein kinase C, was employed to investigate the role of this enzyme in the elaboration of neurites in PC12 pheochromocytoma cells. Normally, PC12 cells respond to NGF by morphologically differentiating into sympathetic neuron-like cells, exhibiting a marked hypertrophy, and extending slender neurites piloted by well defined growth cones. The elaboration of NGF-induced neurites was found to be reversibly inhibited by sphingosine in a dose-dependent manner (IC50 = 2.5-5 microM), while similar concentrations of several structural analogs were inactive. The suppression of neurite outgrowth by sphingosine was antagonized by the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA), which binds to and directly activates protein kinase C. In the presence of NGF, TPA treatment increased the incidence of neurite outgrowth, and this increase, in turn, was antagonized by sphingosine. The binding of [3H]phorbol 12,13-dibutyrate to specific phorbol ester binding sites in PC12 cells was inhibited by sphingosine at concentrations similar to those which inhibited neurite outgrowth. The effects of sphingosine on TPA-directed protein phosphorylation were examined in situ, revealing inhibition of [32P]phosphate incorporation into cellular proteins. The specific TPA-directed phosphorylation of tyrosine hydroxylase was inhibited by sphingosine, as was the resulting increase in enzyme activity. The effects of sphingosine on the levels of alpha- and beta-tubulin mRNAs were also examined in an effort to delimit the locus of protein kinase C action. Concentrations of sphingosine which suppressed neurite outgrowth did not inhibit the NGF-directed elevation of tubulin transcript levels. Taken together, these results reveal the presence of a sphingosine-sensitive pathway in neurite outgrowth and indicate that protein kinase C plays a role in mediating the neuritogenic effects of NGF. Furthermore, the results suggest that protein kinase C acts at a distal segment of the neurite growth pathway.
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PMID:Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C. 316 37

N-Methyl-4-phenylpyridinium ion (MPP+), a reaction product of a neurotoxin, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was found to inhibit aromatic L-aminoacid decarboxylase activity in rat clonal pheochromocytoma PC12h cells. The enzyme activity was enhanced to several folds by addition of a cofactor, pyridoxal phosphate, and MPP+ inhibited the enhancement of the activity by exogenously added pyridoxal phosphate. The inhibition was competitive to pyridoxal phosphate, and the Ki value of MPP+ was 26.7 +/- 0.4 microM, while the Km value of pyridoxal phosphate was 0.645 +/- 0.053 microM. The inhibition was partly irreversible. The enzyme sample was incubated with MPP+ and then dialyzed against phosphate buffer. After dialysis, the inhibited enzyme activity was only partly recovered by addition of pyridoxal phosphate, even though MPP+ was completely removed. Activity of other enzymes, tyrosine hydroxylase and monoamine oxidase could be recovered by dialysis. On the other hand, MPP+ did not affect the binding of the enzyme with the substrate, L-DOPA or 5-hydroxytryptophan.
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PMID:Inhibition of aromatic L-aminoacid decarboxylase in clonal pheochromocytoma PC12h cells by N-methyl-4-phenylpyridinium ion (MPP+). 325 44

Four patients in three families with "peripheral" tetrahydrobiopterin deficiency were investigated. They were characterized biochemically by a tetrahydrobiopterin-responsive hyperphenylalaninaemia, a high neopterin/biopterin ratio in urine and plasma, and normal or elevated concentrations of biopterin, homovanillic acid, and 5-hydroxyindole acetic acid in cerebrospinal fluid. From measurements of the activity of erythrocyte 6-pyruvoyl tetrahydropterin synthase (PTS, formerly called phosphate-eliminating enzyme) and phenylalanine loading tests in the patients and their parents, one patient was demonstrated to be heterozygous for PTS deficiency. The others were obviously genetic compounds (allelism) with incomplete PTS deficiency. Three of the children developed normally, two of them under treatment with tetrahydrobiopterin. In the latter two patients, significantly lower concentrations of biopterin, homovanillic acid, and 5-hydroxyindole acetic acid in cerebrospinal fluid were noted at age 7 months (when treatment was interrupted) than those observed at 3 and 5 weeks, respectively. The infant who is heterozygous for PTS deficiency was born small for gestational age and showed a moderately delayed psychomotor development. It is concluded that "peripheral" tetrahydrobiopterin deficiency is caused by a partial PTS deficiency with sufficient activity to cover the tetrahydrobiopterin requirement of tyrosine 3-hydroxylase and trytophan 5-hydroxylase in brain but not enough for phenylalanine 4-hydroxylase in liver. For therapy, tetrahydrobiopterin, 2-5 mg/kg in a single oral dose per day, is recommended to keep plasma phenylalanine normal. A careful observation of the mental development is indicated.
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PMID:"Peripheral" tetrahydrobiopterin deficiency with hyperphenylalaninaemia due to incomplete 6-pyruvoyl tetrahydropterin synthase deficiency or heterozygosity. 329 9

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.
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PMID:Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells. 358 24


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