Biomedical subjects

P Aroca

Publications and source records attributed to P Aroca.

26 records · Page 2Linked to original sources

PubMed1991

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↗

PubMed1991

After dopachrome.

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Indolequinones↗

PubMed1990

Regulation of mammalian melanogenesis. I: Partial purification and characterization of a dopachrome converting factor: dopachrome tautomerase.

A protein that catalyzes the decoloration of dopachrome has been partially purified from B16 mouse melanoma tumors. The enzyme is preferentially associated to the melanosomes, but it is also found in the microsomal and cytosolic fractions of cellular homogenates. The protein is clearly different from tyrosinase, and should be related to the dopachrome oxidoreductase (Barber et al. (1984) J. Invest. Dermatol. 83, 145-149) and the dopachrome conversion factor (Korner and Pawelek (1980) J. Invest. Dermatol. 75, 192-195) since the reaction product of dopachrome conversion is 5,6-dihydroxyindole-2-carboxylic acid. The protein appears to have an oligomeric structure, with a molecular mass slightly higher than 300 kDa estimated by gel filtration, whereas the molecular mass of the monomer might be approx. 46 kDa estimated by SDS-PAGE electrophoresis. Its Km for dopachrome is around 100 microM. The enzyme is competitively inhibited by indoles and is unaffected by metal chelators. It also has the ability to increase the amount of melanin formed from L-tyrosine by melanoma tyrosinase, and therefore, cannot be considered an 'indole blocking factor' as was suggested for the related dopachrome oxidoreductase. Since the reaction catalyzed by the enzyme is a tautomeric shift on dopachrome, we would propose dopachrome tautomerase (EC 5.3.2.3) as the most precise and informative name.

Animals↗

PubMed1990

Regulation of mammalian melanogenesis. II: The role of metal cations.

Melanogenesis can be divided into two phases. The first one involves two tyrosinase-catalyzed oxidations from tyrosine to dopaquinone and a very fast chemical step leading to dopachrome. The second phase, from dopachrome to melanin, can proceed spontaneously through several incompletely known reactions. However, some metal transition ions and protein factors different from tyrosinase might regulate the reaction rate and determine the structure and relative concentrations of the intermediates. The study of the effects of some divalent metal ions (Zn, Cu, Ni and Co) on some steps of the melanogenesis pathway has been approached using different radiolabeled substrates. Zn(II) inhibited tyrosine hydroxylation whereas Ni(II) and Co(II) were activators. Ni(II), Cu(II) and Co(II) accelerated chemical reactions from dopachrome but inhibited its decarboxylation. Dopachrome tautomerase also decreased decarboxylation. When metal ions and this enzyme act together, the inhibition of decarboxylation was greater than that produced by each agent separately, but amount of carboxylated units incorporated to the melanin was not higher than the amount incorporated in the presence of only cations. The amount of total melanin formed from tyrosine was increased by the presence of both agents. The action of Zn(II) was different from other ions also in the second phase of melanogenesis, and its effect on decarboxylation was less pronounced. Since tyrosine hydroxylation is the rate-limiting step in melanogenesis, Zn(II) inhibited the pathway. This ion seems to be the most abundant cation in mammalian melanocytes. Therefore, under physiological conditions, the regulatory role of metal ions and dopachrome tautomerase does not seem to be mutually exclusive, but rather complementary.

Animals↗

PubMed1990

A new spectrophotometric assay for dopachrome tautomerase.

The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopachrome into 5,6-dihydroxyindole. The advantages of these methods as compared to the previously used procedures are discussed.

Animals↗

PubMed1990

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei).

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.

Animals↗

PubMed1989

A reexamination of the melanin formation assay of tyrosinase and an extension to estimate phaeomelanin formation.

This paper presents some modifications of the melanin formation assay for tyrosinase from the point of view of both eu- and phaeomelanosynthesis. On the one hand, eumelanosynthesis can be estimated using neutral paper filters, such as the 3MM Whatman filters so far employed. The main advantages of this sort of paper are the very low blank values obtained in the absence of tyrosinase and its greater mechanical resistance in the successive washing steps. It is shown that the sensitivity of the assay can be enhanced by the addition of 1 mM Ni(II) to the incubation mixture or of NaOH to stop the enzymatic reaction and allow the incorporation of indolic intermediates into the polymer. Furthermore, the accuracy is also enhanced by the proposed modifications, since all reactions from dopaquinone are standardized, and the assay becomes only dependent on the tyrosinase activity. On the other hand, phaeomelanosynthesis cannot be estimated using neutral paper because of the slow rate of polymerization of the intermediates and the poor absorption of thiol-dopa conjugates to this kind of paper. It is shown that synthesis of this type of melanin can be estimated in the presence of glutathione by means of a cationic filter paper and by washing the excess of the radioactive substrate with distilled water instead of acidic media. Thus, the assay may be adapted to measure eu- or phaeomelanosynthetic activity by introducing slight modifications. This assay must be used with caution if detergent-solubilized tyrosinase is used, because detergents strongly inhibit melanin absorption to paper filters.

Animals↗

PubMed1988

The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway.

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.

Animals↗