Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.
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PMID:Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells. 149 75

It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the hydrolytic treatment becomes lower. Thus total deglycosylation with peptide N-glycosidase F directly provokes an inactivation of DCT. The native enzyme also shows a strong affinity for concanavalin A-Sepharose. This affinity decreases after treatment with neuraminidase and/or beta-mannosidase. The DCT associated with coated vesicles seems to be mostly glycosylated, since the action of glycosylases on the enzyme obtained from these vesicles produced a similar stimulation to that with the melanosomal enzyme. Treatment of cultured melanocytes with tunicamycin elicited a decrease in the amount of active DCT inside the cells. All data suggest that the structure of the carbohydrate moiety of DCT should be very similar to, if not identical with, the structure proposed for tyrosinase by Ohkura, Yamashita, Mishima & Kobata (1984) Arch. Biochem. Biophys. 235, 63-77.
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PMID:The action of glycosylases on dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase. 159 91

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60 degrees C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.
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PMID:Preparation of purified tyrosinase devoid of dopachrome tautomerase from mammalian malignant melanocytes. 823 1

Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on similarity between the protein sequences of the TRPs suggest the presence of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content in purified DCT preparations show the presence of around 1.5 Zn atoms/molecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution experiments of tautomerase activity in the presence of different ions confirmed that the metal cofactor for the DCT active site is zinc. Our results are consistent with Zn2+ chelation by the highly conserved histidine residues homologous to the histidines at the classical copper-binding sites in tyrosinase. This finding accounts for the reaction catalysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidations catalysed by tyrosinase. Based on the predicted tetrahedrical coordination of the zinc ions in the enzyme active site, a molecular mechanism for the catalysis of L-dopachrome tautomerization is proposed. From the present data, the existence of additional ligands for metal ions other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such sites exist, they could be subsidiary binding sites, whose function would be likely to stabilize the protein.
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PMID:Molecular mechanism for catalysis by a new zinc-enzyme, dopachrome tautomerase. 857 77

The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is present in the dermal and epidermal layers of normal skin [Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) J. Invest. Dermatol. 100, 674-680]. Its local concentrations are modified by several stimuli, including wound healing and ultraviolet irradiation. Moreover, TNF-alpha inhibits melanogenesis in normal melanocytes [Swope, V., Abdel-Malek, Z., Kassem, L. & Norlund, J. (1991) J. Invest. Dermatol. 96, 180-185], and is, therefore, a potential autocrine/paracrine regulator of pigmentation. We have analyzed the mechanisms of this effect using B16/F10 melanoma cells as a model. Nanomolar concentrations of TNF-alpha inhibit the tyrosine hydroxylase and dopa oxidase activities of B16/F10 melanocytes, to less than 30% control levels, without effects on tyrosinase-related protein 2/dopachrome tautomerase (TRP2/DCT). The 50% inhibition was obtained at 1 nM TNF-alpha and 48 h treatment. The effect of TNF-alpha was noticeable after 6 h treatment, and maximal after 24 h. This inhibition is explained by decreased intracellular levels of tyrosinase and tyrosinase-related protein 1 (TRP1), but not of TRP2/DCT as detected by Western blotting. Northern-blot experiments showed that the inhibitory effect is partially explained by a reduced accumulation of the corresponding mRNAs, that dropped to about 50% of control values (48 h treatment, 5 nM TNF-alpha). Moreover, the tyrosine hydroxylase and dopa oxidase activities decreased more rapidly in TNF-alpha-treated cells than in control cells, under conditions of inhibition of protein synthesis. This suggests a TNF-mediated reduction of tyrosinase half-life. However, the possibility of an inhibitory post-translational modification of the enzyme induced by TNF cannot be ruled out. Therefore, the inhibitory effect of TNF-alpha on tyrosinase and TRP-1 results from combined effect on mRNA levels and enzymatic activity or protein stability.
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PMID:Mechanisms of melanogenesis inhibition by tumor necrosis factor-alpha in B16/F10 mouse melanoma cells. 969 12

Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.
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PMID:Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin. 1127 91

This study describes an in utero approach for overexpressing genes in a cell-type directed manner. It uses an avian leukosis retroviral expression system coupled with a transgenic mouse line expressing the viral receptor tv-a from a tissue-specific promoter (RCAS-TVA system) (Federspiel et al., 1994, and reviewed in Fisher et al., 1999). A transgenic mouse line was generated expressing tv-a from the Dopachrome tautomerase promoter (DCT-tv-a) in embryonic melanocyte precursors (melanoblasts). RCAS virus encoding beta-galactosidase (RCAS-LacZ) or tyrosinase (RCAS-Tyr) was injected in utero into embryonic day 12.5 albino (tyrosinase inactive) mouse embryos. Animals were analyzed for beta-galactosidase activity or tyrosinase activity (hair pigmentation). RCAS gene expression was detected in 44% and 25% of the transgenic mice, respectively. We demonstrate the RCAS-TVA system coupled with the DCT-tv-a line of mice can be used for in utero infection.
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PMID:In utero complementation of a neural crest-derived melanocyte defect using cell directed gene transfer. 1141 66

Tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1/gp75) and dopachrome tautomerase (DCT/TYRP2) belong to a family of melanocyte-specific gene products involved in melanin pigmentation. During melanocyte development expression of tyrosinase family genes is thought to be orchestrated in part by the binding of a shared basic helix-loop-helix transcription factor MITF to the M box, a regulatory element conserved among these genes. In transformed melanocytes, expression of tyrosinase and TYRPs is highly variable. Whereas TYR expression in melanoma cells is regulated by both transcriptional and post-translational mechanisms, TYRP1/gp75 transcription is often completely extinguished during melanoma tumor progression. In this study, we investigated the mechanisms of selective repression of TYRP1 transcription. Interestingly, in early stage melanoma cells TYRP1 mRNA could be induced by inhibition of protein synthesis. Transient transfection experiments with a minimal TYRP1 promoter showed that the promoter activity correlates with expression of the endogenous TYRP1 gene. Nucleotide deletion analysis revealed novel regulatory sequences that attenuate the M box-dependent MITF activity, but which are not involved in the repression of TYRP1. Gel mobility shift analysis showed that binding of the transcription factor MITF to the TYRP1 M box is selectively inhibited in TYRP1(-) cells. These data suggest that protein factors that modulate the activity of MITF in melanoma cells repress TYRP1 and presumably other MITF target genes.
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PMID:Selective down-regulation of tyrosinase family gene TYRP1 by inhibition of the activity of melanocyte transcription factor, MITF. 1213 92

Ultraviolet radiation stimulates pigmentation in human skin, but the mechanism(s) whereby this increase in melanin production (commonly known as tanning) occurs is not well understood. Few studies have examined the molecular consequences of UV on human skin of various racial backgrounds in situ. We investigated the effects of UV on human skin of various races before and at different times after a single 1 minimal erythemal dose UV exposure. We measured the distribution of DNA damage that results, as well as the melanin content/distribution and the expression of various melanocyte-specific genes. The density of melanocytes at the epidermal:dermal junction in different types of human skin are remarkably similar and do not change significantly within 1 wk after UV exposure. The expression of melanocyte-specific proteins (including TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (tyrosinase-related protein 2), MART1 (melanoma antigens recognized by T-cells) gp100 (Pmel17/silver), and MITF (micropthalmia transcription factor)) increased from 0 to 7 d after UV exposure, but the melanin content of the skin increased only slightly. The most significant change, however, was a change in the distribution of melanin from the lower layer upwards to the middle layer of the skin, which was more dramatic in the darker skin. These results provide a basis for understanding the origin of different skin colors and responses to UV within different races.
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PMID:Mechanisms of skin tanning in different racial/ethnic groups in response to ultraviolet radiation. 1595 11

Most of the melanoma markers used today are melanocytic markers or pigmentation pathway-associated genes driven by the microphthalmia transcription factor, MITF, and include among others, tyrosinase, dopachrome tautomerase, DCT, melan-A and S100B. Genomic studies repeatedly revealed several novel melanoma marker genes including those of the transcription factor NOTCH2, WNT5A, proliferation-associated genes TOPO2A and CDC2, membrane receptors FGFR and EphA3, adhesion molecules N-cadherin, beta3 integrin and syndecan-4, and the cell surface antigens CD59/protectin and MIA. Other genomic analyses tried to define the gene signature of the metastatic disease but failed to find a consistent one except the gold standard genes of beta3 integrin, syndecan-4 and WNT5a. Studies on the gene signatures of chemoresistance and cytokine sensitivity of melanoma clearly defined apoptosis-resistance as one of the key elements of the above biological properties, but the data are controversial, mostly because of the use of inappropriate model systems and the lack of confirmation on clinical samples. Accordingly, application of genomic technologies must be more "translational" to provide breakthrough in melanoma diagnosis and therapy.
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PMID:Melanoma genomics reveals signatures of sensitivity to bio- and targeted therapies. 1743 76


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