Is dopachrome tautomerase necessary to get DHICA from dopachrome?
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Hydrogen-Ion Concentration↗
Biomedical subjects
F Solano
Publications and source records attributed to F Solano.
Tyrosinase isoenzymes: two melanosomal tyrosinases with different kinetic properties and susceptibility to inhibition by calcium.
Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS-PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and dopa oxidase activity and were stereospecific and sensitive to phenylthiourea, providing that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme. Immunoprecipitation and Western blots performed with the specific antibody alpha PEP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were noncompetitively inhibited by Ca2+ at physiologically relevant concentrations. However, the b protein was apparently more susceptible, since maximal inhibition was reached at lower Ca2+ concentrations for LEMT. Moreover, binding of Ca2+ to the tyrosinases resulted in a noticeable thermal destabilization of the enzymes, which was also more pronounced for LEMT.
Animals↗
The DHICA oxidase activity of the melanosomal tyrosinases LEMT and HEMT.
Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Tyrosinase isoenzymes in mammalian melanocytes. 2. Differential activation by alpha-melanocyte-stimulating hormone.
In mouse melanoma melanocytes, alpha-melanocyte-stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH, or its superpotent analog [Ahx4, DPhe7]MSH promotes a larger induction of tyrosine hydroxylase than of dopa oxidase activity in whole cell extracts. This higher activation of tyrosine hydroxylation was found not only in the melanosomal but also in the microsomal fraction; it appears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase activity of control and theophylline-treated extracts displayed several kinetic differences, including different Km values for both substrates and requirements for the cofactor L-dopa. SDS/PAGE, followed by a sensitive specific activity stain, demonstrated that melanosomes of control cells contain one lower-electrophoretic-mobility form of tyrosinase, whereas melanosomes of cells treated with either theophylline or MSH display, in addition to the lower-mobility form, a faster-migrating activity band. These tyrosinase forms are not interconvertible by proteolysis or deglycosylation. Their nature is discussed as related to the properties of the previously described low- and high-electrophoretic-mobility tyrosinases (LEMT and HEMT), as well as of the proteins encoded by the c and b loci.
Animals↗
Tyrosinase isoenzymes in mammalian melanocytes. 1. Biochemical characterization of two melanosomal tyrosinases from B16 mouse melanoma.
B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Km for 3,4-dihydroxy-L-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of Km for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L-dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2-phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.
Animals↗
Sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis by the protein kinase C inhibitor staurosporine.
This study shows the sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis without modifying intracellular free calcium concentrations. The culture of cells in the absence of serum during 6 h failed to produce spontaneous DNA fragmentation. However, when cultures were carried out in the presence of the putative protein kinase C inhibitor staurosporine (1 microM), cells underwent apoptosis. Tumor-promoting phorbol ester failed to inhibit this effect. The presence of the chemotherapeutic drug bleomycin (0.6 mg/ml) in the culture medium reproduced the same pattern of cleaved DNA in nucleosomal pieces. Lower doses of staurosporine (0.1-1 nM) inhibited DNA synthesis but were unable to trigger apoptosis in 6 h culture. Higher doses of staurosporine (0.1-1 microM), which abolished DNA synthesis almost completely, were needed to trigger apoptosis. Short incubation of RIN m5F cells in the presence of staurosporine did not produce any change in the concentration of intracellular calcium or in the integrity of the plasma membrane. These results suggest the involvement of protein kinase C in RIN m5F cell survival.
Alkaloids↗
Glutathione depletion increases tyrosinase activity in human melanoma cells.
The aim of the present work was to estimate the effect of intracellular glutathione depletion on melanogenesis in human melanoma cells. We determined tyrosine hydroxylation activity, the rate-limiting step of the pathway, and 14C-melanin formation, an assay reflecting the global eumelanogenic pathway. Intracellular glutathione was depleted by treatment with buthionine-S-sulfoximine, a well-known inhibitor of gamma-glutamylcysteine synthetase. The intracellular depletion of glutathione was substantial after 20 h of incubation with 50 microM buthionine-S-sulfoximine, although a significant effect could be observed after 6 h. Tyrosine hydroxylase activity increased in parallel with glutathione depletion, to reach 160% with respect to the control values during 24 h of buthionine-S-sulfoximine treatment. We have found the response to buthionine-S-sulfoximine to be dose dependent and the two different human cell lines HBL and LND1 to have similar, if not identical, responses. 14C-melanin formation assay revealed even greater activation, up to 400% of the control values. This indicates that glutathione depletion may have two distinct effects: first, a direct one on tyrosinase activity and, second, an effect on the promotion of eumelanogenesis. The stimulation of tyrosine hydroxylase can be explained by a possible inactivation of the enzyme by endogenous thiol compounds rather than by a direct effect of buthionine-S-sulfoximine itself on tyrosinase. The data suggest that thiol compounds may play a role for stimulation of melanogenesis by ultraviolet radiation.
Antimetabolites, Antineoplastic↗
Preparation of purified tyrosinase devoid of dopachrome tautomerase from mammalian malignant melanocytes.
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.
Animals↗
Improved tyrosinase activity stains in polyacrylamide electrophoresis gels.
Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational processing with possible regulatory implications. So far, SDS-PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However, the relatively poor sensitivity of the currently available specific activity stain based on incubation of the gels with L-dopa until the formation of melanin has severely limited the use of electrophoresis in regulation studies. Two alternative staining procedures are presented and discussed. The first one involves the fluorographic detection of radioactive melanin after incubation of the gels in the presence of L-[3-14C]-dopa. A similar method has already been used by others (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84-89), but its performance has not yet been compared to the one of the dopa procedure. The sensitivity of this method can be varied by adjusting the isotopic dilution of the tracer and/or the time of exposure of the gel, but it is at least ten times higher than the one of the colorimetric stain. Moreover, the intensity of the bands is proportional to the initial tyrosinase activity over a wide range. Using this procedure, the activity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the formation of a colored adduct between dopaquinone and MBTH.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils.
The oxidation of free coelenterazine by superoxide anion was analyzed and compared to the oxidation by the semisynthetic photoprotein obelin, prepared by incorporation of synthetic coelenterazine into apoobelin. The oxidation of bound coelenterazine was triggered upon binding of calcium to the reconstituted photoprotein. The oxidation of free synthetic coelenterazine, in the absence of the apoprotein, was triggered by superoxide anion. The production of reactive oxygen metabolites by fMet-Leu-Phe- and 4b-phorbol 12b-myristate 13a-acetate-stimulated neutrophils was studied by means of the luminescence of synthetic coelenterazine. The features of this chemiluminescent probe were compared with those of luminol and are summarized as follows: (a) coelenterazine-dependent chemiluminescence was inhibited by superoxide dismutase; (b) coelenterazine was as sensitive as luminol in detecting the oxidative burst of neutrophils; (c) azide failed to inhibit coelenterazine chemiluminescence; (d) in contrast with luminol, which requires the catalytic removal of hydrogen peroxide, coelenterazine chemiluminescence did not depend on the activity of cell-derived myeloperoxidase. These results indicate the usefulness of coelenterazine as a very sensitive and specific chemiluminescence probe of superoxide anion.
Aequorin↗
Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.
The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.
Animals↗
Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells. Evidence for a differential induction of two distinct isoenzymes.
Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.
Animals↗
The action of glycosylases on dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase.
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.
Animals↗
Proteolysis with trypsin of mammalian tyrosinase isoforms from B16 mouse melanoma.
In spite of the central role of tyrosinase in mammalian pigmentation, few data are available on its structure and structure-function relationships based on direct analysis of the protein. A number of reasons have been invoked to account for this situation, including the problems for its purification and its resistance to proteases. However, no study on the effects of proteases on purified tyrosinase has been reported. We have purified the melanosomal and cytosolic tyrosinases from B16 mouse melanoma and analyzed their susceptibility to trypsin digestion. Both isoforms are sensitive to trypsin, and display similar peptide maps and kinetics of proteolysis, suggesting that they are products of the same gene. The peptide maps and the kinetics of appearance of the fragments were consistent with the sequential removal of N-terminal peptides, leading to a core of 55.3 kDa for the melanosomal form and 48.6 kDa for the cytosolic enzyme. This core was apparently resistant to further proteolysis and catalytically inactive. The difference in molecular weight for the core of the cytosolic and melanosomal forms is the same as that calculated for the native isoforms. The kinetics of enzyme inactivation indicate that the tyrosine hydroxylase and Dopa oxidase activities of tyrosinase are lost at the same rate, and should therefore display similar if not identical structural requirements. The results are discussed in terms of the relationship of both isoforms and of the putative protein sequences deduced from the cDNA clones proposed for tyrosinase.
Animals↗
Alpha-MSH and other melanogenic activators mediate opposite effects on tyrosinase and dopachrome tautomerase in B16/F10 mouse melanoma cells.
alpha-MSH was found to decrease the recently characterized dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of the agent and the time of exposure. In all cases, both melanin production and tyrosinase activity were activated by these treatments, although the grade of tyrosine hydroxylase and dopa oxidase stimulation was different. Moreover, no correlation among the intensities of dopachrome tautomerase inhibition and tyrosinase activation by the tested agents could be obtained. The significance of these results in the regulation of mammalian melanogenesis is discussed.
Animals↗
Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.
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.
Amphotericin B↗
Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome.
A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optimal pH around 6.8. By contrast, the kinetics of dopachrome rearrangement catalysed by cupric ions are not dependent on the stereochemistry of the substrate, are affected by EDTA and are not influenced by the pH of the medium in the range between 5-7.5. Both cupric ions and DCT catalyse the rearrangement of dopachrome to give 5,6-dihydroxyindole-2-carboxylic acid (DICA) rather than 5,6-dihydroxyindole (DI). However, at comparable activity, the ratio of formation DICA/DI is significantly higher in the enzyme-catalysed than in the metal-catalysed reaction. These results provide an improved background to look into the mode of action of DCT and metal ions, enabling a clear cut differentiation between the effects of the two factors when both are present in biological extracts.
Animals↗
Specificity of dopachrome tautomerase and inhibition by carboxylated indoles. Considerations on the enzyme active site.
Dopachrome tautomerase (EC 5.3.2.3) catalyses the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) within the melanin-formation pathway. We have analysed a series of substrate analogues and related compounds as possible substrates and inhibitors of tautomerization. The enzyme appears to be highly specific since D-dopachrome, alpha-methyldopachrome, dopaminochrome, adrenochrome methyl ether and deoxyadrenochrome are not substrates. Conversely, dopachrome tautomerase catalyses the tautomerization of dopachrome methyl ester, suggesting that a carboxy group, either free or as a methyl ester, is essential for enzyme recognition. No inhibition of dopachrome tautomerization was observed in the presence of either semiquinonic compounds, such as tropolone and L-mimosine, or pyrrole-2-carboxylic acid and unsubstituted indole. However, a number of indole derivatives, including DHICA, the product of dopachrome tautomerization, and the analogues 5-hydroxyindole-2-carboxylic and indole-2-carboxylic acid were able to inhibit the enzyme. Furthermore, indoles with a side chain at position 3 of the ring and containing a carboxylic group at the gamma-position of this chain, such as L-tryptophan or indole-3-propionic acid, are stronger inhibitors of the enzyme. Indole-3-carboxylic acid, indole-3-acetic acid and indole-3-butyric acid are very weak inhibitors, showing that the carboxylic group needs to be located at an optimal distance from the indole ring to mimic the carboxylic group at position 2 on the authentic substrate.
Ammonium Sulfate↗