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

A method for measuring the dopa oxidase (DO) activity of human hair bulb tyrosinase has been developed and the results of this method have been compared with the tyrosine hydroxylase (TH) activity of hair bulb tyrosinase for brown-, black-, blond-, and red-haired subjects. The method takes advantage of the rapid trapping of dopaquinone by cysteine with the subsequent formation of cysteinyldopas which can be measured by high-performance liquid chromatography. 5-S-Cysteinyldopa (5SCD) and 2-S-cysteinyldopa (2SCD) were detected in the reaction products. Formation of 5SCD correlated with the TH activity over the full range of hair colors and enzyme activity, while 2SCD appeared to be formed nonenzymatically. The absolute amount of 2SCD was constant for each individual but did not correlate with hair color or TH activity. The formation of 5SCD was linear for 60 min while most of the 2SCD was formed within seconds and did not change with time. White hair bulbs which demonstrated no TH activity formed 2SCD, but not 5SCD. We conclude that tyrosinase activity can be quantitated in human hair bulbs by this method, and that TH and DO are coordinate functions of tyrosinase over a broad range of hair color and enzyme activity.
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PMID:Dopa oxidase activity in human hair bulbs measured by high-performance liquid chromatography. 287 16

The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.
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PMID:The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway. 290 72

The acetone precipitation of a partially purified tyrosine 3-monooxygenase (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) resulted in the complete loss of enzymatic activity. The enzymatic activity was restored by incubation with iron and dithiothreitol. The restoration of the activity was a pH-, temperature- and time-dependent reaction. Since cobalt, nickel, copper, zinc, manganese, cadmium, magnesium calcium and barium ions were all ineffective in restoring activity, iron ion appeared to be specifically required in the restoration of the enzyme activity. Dithiothreitol could be partially replaced in the restoration step by glutathione, 2-mercaptoethanol or cysteine.
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PMID:Inactivation of tyrosine 3-monooxygenase by acetone precipitation and its restoration by incubation with a sulfhydryl agent and iron. 611 46

Oxidative stress has been linked to the destruction of dopaminergic neurons in the substantia nigra and may be a significant factor in both Parkinson's disease and MPTP toxicity. Using primary cultures of embryonic rat mesencephalon and standard immunocytochemical techniques, we have examined the survival of tyrosine hydroxylase-containing (TH+) neurons cultured in the presence of antioxidants and/or in an environment of low oxygen partial pressure. The number of TH+ neurons increased approximately twofold if superoxide dismutase, glutathione peroxidase (GP), or N-acetyl cysteine (NAC) were added to the culture media. Exposure of the neurons to a 5% oxygen environment (38 torr, i.e., 38 mm Hg) also increased the survival of TH+ neurons by about twofold. A dramatic enhancement of survival, however, was seen when NAC was used in combination with the 5% oxygen environment. In this case, the number of TH+ neurons increased fourfold from nontreated controls. Morphological changes were also noted. GP increased the average neurite length while NAC increased the average area of the cell body in the TH+ neuron. These results suggest that manipulation of oxidative conditions by changing the ambient O2 tension or the level of antioxidants promotes survival of TH+ neurons in culture and may have implications for transplantation therapies in Parkinson's disease.
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PMID:Protection from oxidation enhances the survival of cultured mesencephalic neurons. 772 Aug 26

1. Tryptophan has been shown to inhibit dopa-oxidation by melanosomal tyrosinase. 2. The inhibition is of mixed-type with Ki = 1.6 x 10(-3) M. 3. Tryptophan does not interact with the oxidation product of the dopa-oxidase reaction. 4. Neither oxygen nor hydroxyl radicals are involved in the inhibition found in presence of tryptophan. 5. Tryptophan, like dopa, also inhibits tyrosine hydroxylase and dopa-oxidase activity of melanosomal tyrosinase and its inhibitory mechanism differs from inhibition due to non-substrate type compounds like cysteine, ascorbic acid. 6. These experiments together with previous findings suggest that the status of tryptophan may be similar to that of dopa in relation to regulation of melanogenesis.
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PMID:The effect of tryptophan on dopa-oxidation by melanosomal tyrosinase. 822 74

Melanocytic cells can produce two types of pigment, pheomelanin or eumelanin. We used two types of human melanoma cell lines to explore the regulation of pigmentation by biochemical and enzymatic studies. These two cell lines were previously designated as either pheomelanotic or of mixed type when cultured in a medium rich in cysteine. We analyzed the effects of L-cysteine depletion on melanin synthesis and the involvement of the tyrosinase-related proteins in the production of both eumelanin and pheomelanin. Cultures were exposed to L-cysteine concentrations ranging from 206 to 2.06 microM, and the following parameters were measured: tyrosine hydroxylase activity, intracellular L-cysteine and glutathione concentrations, eumelanin and pheomelanin formation, and tyrosinase-related protein-1 and -2 mRNA levels. Extracellular L-cysteine depletion significantly increased tyrosine hydroxylase activity and promoted both eumelanogenesis and visible pigmentation in both lines. In contrast, pheomelanogenesis was increased only in the pheomelanotic cell line. Whereas eumelanogenesis was apparent upon L-cysteine depletion, tyrosinase-related protein-1 expression was not induced in the pheomelanotic cells, and tyrosinase-related protein-2 expression remained unchanged. Thus, tyrosinase-related protein-1 mRNA expression seems to be concomitant with eumelanogenesis when the L-cysteine concentration is high, but does not appear essential for eumelanogenesis at low L-cysteine concentrations. The mechanisms governing pheomelanin to eumelanin balance are dependent on L-cysteine, glutathione, and tyrosinase-related protein-1 expression, but none of these factors alone appears to be dominant in directing the synthesis of a particular type of melanin.
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PMID:Cysteine deprivation promotes eumelanogenesis in human melanoma cells. 887 52

In the presence of thiols, tyrosine hydroxylase (TH) oxidizes L-dihydroxyphenylalanine (L-DOPA) with a specific activity of up to 140 nmol min(-1) mg(-1) at 37 degrees C and pH 7.0, which is approximately 12-50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2-mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of L-DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.
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PMID:L-DOPA is a substrate for tyrosine hydroxylase. 932 1

Neurite extension of PC12 cells induced by nerve growth factor (NGF) is a well-known model of neuronal differentiation. In this study, the incubation of PC12 cells in a 50% O2 atmosphere (hyperoxia) caused neurite extension. In these cells, amounts of differentiation-marker proteins, tyrosine hydroxylase and neurofilament M increased. The effects of hyperoxia were inhibited by ascorbic acid or N-acetyl-cysteine, antioxidant reagents, suggesting the involvement of reactive oxygen species (ROS). In support of this, artificial generation of free radicals induced the same effects as hyperoxia. In these cells, total phosphorylation of cellular proteins was enhanced similar to NGF-treated cells. These results suggest that hyperoxia enhances the signal for neuronal differentiation by producing ROS, resulting in the induction of the differentiated neuronal phenotype of PC12 cells.
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PMID:Hyperoxia induces the differentiated neuronal phenotype of PC12 cells by producing reactive oxygen species. 942 74

Glutathione (GSH) and cysteine (CysH) have both been implicated in the biogenesis of the pheomelanin precursor 5-S-cysteinyldopa (5-S-CD). However, recent studies have shown that only CysH is transported across the membrane of isolated melanosomes, and that the positive regulation of CysH in pigment cells leads to an increased production of 5-S-CD. In the present study, the question was examined as to whether melanin precursors and tyrosinase could be coregulated by cellular thiols. To address this issue, the levels of CysH and GSH were varied in normal melanocytes and melanoma cells using buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis. Treatment with 50-100 microM BSO decreased GSH levels to less than 10% of control, and increased CysH levels between two- and five-fold in both cell types. Concomitant with this, an increase in the ratio of 5-S-CD to DOPA and a decrease in the pigment content of the cells were observed. The decrease in cell pigmentation was associated with strong decreases in tyrosine hydroxylase activity and 14C-melanin production. Only melanoma cells showed a modified tyrosinase isozyme pattern on Western immunoblots in response to BSO, while the mRNA expression of tyrosinase and TRP-1 were unchanged in both cell types. These results suggest that the balance between CysH and GSH, which is partly determined by the rate of utilization of CysH for GSH biosynthesis, regulates not only the levels of 5-S-CD and DOPA but also the melanogenic activity of pigment cells. Since DOPA functions as a cofactor in the monophenolase reaction of tyrosinase, it is proposed that the ratio of 5-S-CD to DOPA may be an important factor in the regulation of tyrosinase activity in situ.
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PMID:Co-regulation of melanin precursors and tyrosinase in human pigment cells: roles of cysteine and glutathione. 1064 2

Caspases are cysteine proteases involved in apoptotic cell death, and pharmacological caspase inhibition has been demonstrated to prevent neuronal cell death in certain experimental paradigms. In this study, the role of caspase-1 and -3 in the death of dopaminergic neurons derived from the E14 rat ventral mesencephalon (VM) has been examined in two model systems using peptide caspase inhibitors. First, cell death was induced in vitro by withdrawing serum after 2 days. Different doses of caspase-1 (IL-1beta converting enzyme) and caspase-3 inhibitors (Ac-DEVD-cmk) were added to the medium at the time of serum withdrawal, and the ability of the inhibitors to promote dopaminergic neuronal survival and prevent activation of caspase-3 was assessed at 7 days. Immunostaining using tyrosine hydroxylase (TH) and cleaved caspase-3 antibodies demonstrated that caspase-1 and -3 inhibitors reduce caspase-3 activation as well as overall cell death. This did not, however, improve the survival of TH-positive neurons, although it did appear to promote their maturation. The second paradigm investigated the effects of these inhibitors in the 6-hydroxydopamine rat model of PD, and similarly, addition of caspase-1 or -3 inhibitor during tissue preparation or immediately prior to grafting of VM tissue did not promote dopaminergic neuronal survival. These results demonstrate that the reduction of apoptotic cell death by pharmacological inhibition of caspase-1 and -3 does not increase dopaminergic neuronal survival in these paradigms and suggest either that caspase-3 activation is not the major determinant of dopaminergic neuronal death in vitro and in grafts or that the ability of caspase inhibitors to rescue cells depends upon the degree of apoptotic stress. This implies that strategies to improve dopaminergic cell survival in clinical programmes of transplantation for PD will need to target other pathways of cell death.
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PMID:Death of dopaminergic neurons in vitro and in nigral grafts: reevaluating the role of caspase activation. 1152 Jan 20


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