A Method for Detecting Dopaminechrome Isomerase Activity on Gels

Melanogenesis involves oxidation of 3,4-dihydroxyphenylalanine (dopa) to dopachrome which then is converted into 5,6-dihydroxyindole by dopachrome isomerase. 5,6-Dihydroxyindole is oxidized to its quinone which in turn is metabolized nonenzymatically to melanin. In addition to dopachrome isomerase, a new dopminechrome isomerase activity involved in the conversion of dopaminechrome into 5,6-dihydroxyindole has been observed in the larva of Rhinoceros oryctes. This dopaminechrome isomerase differs from dopachrome isomerase in its electrophoretic mobility and substrate specificity. The present study reports a specific, sensitive and rapid staining method for detecting dopaminechrome isomerase activity after electrophoresis. Using this new method, the presence of the dopaminechrome isomerase activity, which is involved in melanogenesis, could easily be detected by staining tyrosinase embedded native gels in dopamine solution. Tyrosinase entrapped in the gels converts dopamine in dopaminechrome. The dopaminechrome isomerase separated in the gels catalyzes dopaminechrome to 5,6-dihydroxyindole which is oxidized further by tyrosinase to colored melanochrome. The dopaminechrome isomerase appears as a bluish purple band against a pink background.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

The effect of polyamines on tyrosinase activity

The effect of several polyamines on the activity of tyrosinase from different sources has been studied. Diaminoethane, 1,3-diaminopropane and putrescine activated tyrosinase from Harding-Passey mouse melanoma but did not activate frog epidermis or mushroom tyrosinases. 1,3-diaminopropane was the strongest activator (Ka = 0.23 mM). The activation was saturable and dependent on the ionic strength. Cadaverine, 1,6-diaminohexane and spermidine had no effect on any tyrosinase. However, spermine inhibited melanoma tyrosinase more than the mushroom and frog epidermis enzymes. These results show that the effect of polyamines on mammalian tyrosinase is due to direct enzyme-oligoamine interactions rather than to a nonspecific action on L-dopa oxidation products, and suggest that physiological polyamines might play a modulatory role on mammalian melanogenesis.     

Insect melanogenesis. II. Inability of Manduca phenoloxidase to act on 5,6-dihydroxyindole-2-carboxylic acid

Eumelanins in animals are biosynthesized by the combined action of tyrosinase, 3,4-dihydroxyphenylalanine (DOPA)chrome isomerase, and other factors. Two kinds of eumelanins were characterized from mammalian systems; these are 5,6-dihydroxyindole (DHI)-melanin and 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. In insects, melanin biosynthesis is initiated by phenoloxidase and supported by DOPAchrome isomerase (decarboxylating). Based on the facts that DOPA is a poor substrate for insect phenoloxidases and DHI is the sole product of insect DOPAchrome isomerase reaction, it is proposed that insects lack DHICA-melanin. Accordingly, the phenoloxidase isolated from the hemolymph of Manduca sexta failed to oxidize DHICA. Control experiments reveal that mushroom tyrosinase, as well as laccase, which is a contaminant in the commercial preparations of mushroom tyrosinase, are capable of oxidizing DHICA. Neither the whole hemolymph nor the cuticular extracts of M. sexta possessed any detectable oxidase activity towards this substrate. Thus, insects do not seem to produce DHICA-eumelanin. A useful staining procedure to localize DHICA oxidase activity on gels is also presented.     

Enzymatic Synthesis of (+)Catechin-α-glucoside and Its Effect on Tyrosinase Activity

The glycosidation of (+)catechin, which has five hydroxyl groups with cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) and soluble starch has been studied. One of the transfer products was purified and its structure was determined to be (+)catechin 3?-0-α-D-glucopyranoside. This glucoside noncompetitively inhibited the activity of tyrosinase from mushroom, IC₅₀ being 5.8 mM, but didn’t inhibit that from mouse melanoma. In contrast, arbutin (hydroquinone-0-β-D-glucopyranoside) inhibited both tyrosinases.     

Comparative study of tyrosinases from different sources: relationship between halide inhibition and the enzyme active site

The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed.     

A spectrophotometric assay for mammalian tyrosinase utilizing the formation of melanochrome from L-dopa

A simple spectrophotometric method for a rapid determination of tyrosinase (EC 1.14.18.1) is described. The basis of the assay is the incubation of the enzyme with L-dopa in the presence of an optimal concentration of Zn2+ ions and the measurement of the formation of melanochrome, as indicated by the rise in absorbance at 540 nm. Final absorbance change reflects probably two activities of tyrosinase: the oxidation of dopa to dopaquinone and the conversion of 5,6-dihydroxyindole to melanochrome. Using a purified preparation from hamster melanoma, the assay was found to be more sensitive than the commonly used dopachrome assay. Comparison with some other currently available methods for assaying tyrosinase is presented and potential applications of the assay are discussed.     

Bacterial tyrosinases and their applications

Tyrosinases with different physico-chemical properties have been identified from various bacterial phyla such as Actinobacteria and Proteobacteria and their production is often inducible by environmental stresses. Tyrosinases are enzymes catalysing the oxidation of mono- and di-phenolic compounds to corresponding quinones with the concomitant reduction of molecular oxygen to water. Since the quinone produced can further react non-enzymatically with other nucleophiles, e.g. amino groups, many tyrosinases have a recorded cross-linking activity on proteins. Various bacterial tyrosinases oxidise tyrosine, catechol, l/d-DOPA, caffeic acid and polyphenolic substrates such as catechins. This substrate specificity has been exploited to engineer biosensors able to detect even minimal amounts of different phenolic compounds. The physiological role of tyrosinases in the biosynthesis of melanins has been used for the production of coloured and dyeing agents. Moreover, the cross-linking activity of tyrosinases has found application in food processing and in the functionalisation of materials. Numerous tyrosinases with varying substrate specificities and stability features have been isolated from bacteria and they can constitute valuable alternatives to the well-studied tyrosinase from common mushroom.     

Purification of hamster melanoma tyrosinases and structural studies of their asparagine-linked sugar chains

In cultured melanotic melanoma, a marked decrease of pigmentation has been found to be induced by the addition of tunicamycin [Y. Mishima and G. Imokawa (1983) J. Invest. Dermatol. 81, 106-114]. Since it appears that this impaired pigmentation arises from the loss of asparagine-linked sugar chains serving as a signal for transport of tyrosinase from GERL (Golgi-associated endoplasmic reticulum of lysosomes) to premelanosomes, tyrosinases from the membrane fraction of Greene's hamster melanoma have been purified, and the structures of their sugar chains have been analyzed. Two kinds of tyrosinases were purified by Triton X-100 solubilization; DEAE-cellulose, Sephadex G-200, and DEAE-Sephadex column chromatography; and preparative polyacrylamide gel electrophoresis. The two tyrosinases were separated by polyacrylamide gel electrophoresis, and both corresponded to Mr 69,000. Their asparagine-linked sugar chains were released by hydrazinolysis and analyzed. The sugar chains of the two tyrosinases were identical except for the sialic acid contents. One mole of each tyrosinase contained 1 mol of high-mannose-type sugar chains and 3 mol of complex-type sugar chains. The former chain has Man3 approximately 5 X GlcNAc2 and the latter has Man3 X GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their core structures. The complex-type sugar chains are composed of mono-, bi-, tri-, and tetraantennary sugar chains, with +/- Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----as their outer chains.     

Insect Melanogenesis. II. Inability of Manduca Phenoloxidase to Act on 5, 6-Dihydroxyindole-2-Carboxylic Acid1

Eumelanins in animals are biosynthesized by the combined action of tyrosinase, 3, 4-dihydroxyphenylalanine (DOPA)chrome isomerase, and other factors. Two kinds of eumelanins were characterized from mammalian systems; these are 5,6-dihydroxyindole (DHI)-melanin and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. In insects, melanin biosynthesis is initiated by phenoloxidase and supported by DOPAchrome isomerase (decarboxylating). Based on the facts that DOPA is a poor substrate for insect phenoloxidases and DHI is the sole product of insect DOPAchrome isomerase reaction, it is proposed that insects lack DHICA-melanin. Accordingly, the phenoloxidase isolated from the hemolymph of Manduca sexta failed to oxidize DHICA. Control experiments reveal that mushroom tyrosinase, as well as laccase, which is a contaminant in the commercial preparations of mushroom tyrosinase, are capable of oxidizing DHICA. Neither the whole hemolymph nor the cuticular extracts of M. sexta possessed any detectable oxidase activity towards this substrate. Thus, insects do not seem to produce DHICA-eumelanin. A useful staining procedure to localize DHICA oxidase activity on gels is also presented.     

Electrochemical identification of dopachrome isomerase in Drosophila melanogaster

A principal reaction in the eumelanin biosynthetic pathway is the conversion of dopachrome (DC) to dihydroxyindole(s). Dopachrome isomerase (DI), the enzyme that catalyzes this reaction, was detected for the first time in larvae of D. melanogaster. Unlike the enzyme from B16 mouse melanoma cells which converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the insect enzyme forms 5,6-dihydroxyindole (DHI). The activity of the insect DI was linear through 15 min incubation, and the amount of DHI produced was proportional to the amount of enzyme that was incorporated into the reaction mixtures.     

A pulse radiolysis investigation of the oxidation of indolic melanin precursors: evidence for indolequinones and subsequent intermediates

The rate constants associated with the series of successive transient absorptions initiated by one-electron oxidation of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), precursors of melanin, and N-methyl-5,6-dihydroxyindole (NMDHI), a model compound, have been studied by pulse radiolysis. The initial transient species resulting from N3. oxidation reaction at pH 7.3-7.4 are assigned as the corresponding semiquinones. In each case, these radicals decayed, probably by disproportionation, into products most readily monitored in the 400-430 nm region. For DHI, the decay in this region could be fitted by two parent concentration independent first-order processes. These may correspond to transformations between 5,6-indolequinone, and its quinone-imine and quinone-methide tautomers. With NMDHI, on the other hand, a single longer-lived product with a peak around 430 nm predominated after decay of the corresponding radical, due almost certainly to N-methyl-5,6-indolequinone. The data appear to exclude significant melanin polymerisation by condensation of semiquinones, reaction of semiquinones with dihydroxyindoles, self-addition of indolequinones or tautomers, or reaction of indolequinones or tautomers with the parent dihydroxyindoles. It is suggested that polymerisation of melanin may rather occur by stepwise addition of indolequinone methide/imine to reduced oligomeric species.     

Oxyresveratrol and hydroxystilbene compounds. Inhibitory effect on tyrosinase and mechanism of action

Tyrosinase is responsible for the molting process in insects, undesirable browning of fruits and vegetables, and coloring of skin, hair, and eyes in animals. To clarify the mechanism of the depigmenting property of hydroxystilbene compounds, inhibitory actions of oxyresveratrol and its analogs on tyrosinases from mushroom and murine melanoma B-16 have been elucidated in this study. Oxyresveratrol showed potent inhibitory effect with an IC(50) value of 1.2 microm on mushroom tyrosinase activity, which was 32-fold stronger inhibition than kojic acid, a depigmenting agent used as the cosmetic material with skin-whitening effect and the medical agent for hyperpigmentation disorders. Hydroxystilbene compounds of resveratrol, 3,5-dihydroxy-4'-methoxystilbene, and rhapontigenin also showed more than 50% inhibition at 100 microm on mushroom tyrosinase activity, but other methylated or glycosylated hydroxystilbenes of 3,4'-dimethoxy-5-hydroxystilbene, trimethylresveratrol, piceid, and rhaponticin did not inhibit significantly. None of the hydroxystilbene compounds except oxyresveratrol exhibited more than 50% inhibition at 100 microm on l-tyrosine oxidation by murine tyrosinase activity; oxyresveratrol showed an IC(50) value of 52.7 microm on the enzyme activity. The kinetics and mechanism for inhibition of mushroom tyrosinase exhibited the reversibility of oxyresveratrol as a noncompetitive inhibitor with l-tyrosine as the substrate. The interaction between oxyresveratrol and tyrosinase exhibited a high affinity reflected in a K(i) value of 3.2-4.2 x 10(-7) m. Oxyresveratrol did not affect the promoter activity of the tyrosinase gene in murine melanoma B-16 at 10 and 100 microm. Therefore, the depigmenting effect of oxyresveratrol works through reversible inhibition of tyrosinase activity rather than suppression of the expression and synthesis of the enzyme. The number and position of hydroxy substituents seem to play an important role in the inhibitory effects of hydroxystilbene compounds on tyrosinase activity.     

Melanosome proteins duringBufo bufo development

Summary Changes in melanosome protein composition duringBufo bufo development have been investigated. The number of bands in SDS-PAGE varies from stage 7 (sixth cleavage) to stage 19 (heart beat), with a minimum at stages 17 (tail bud) and 19 (heart beat). At least eleven bands appear to be present throughout stages examined. The most relevant difference observed is that three protein bands of 40, 66 and 115 Kdal are expressed only at pre-neurular stages, when only maternal melanosomes are present in the embryo. Moreover, 5,6-dihydroxyindole oxidase activity has been evidenced in the gels, by specific staining. The activity appears to comigrate with tyrosinase.     

Structural modifications in biosynthetic melanins induced by metal ions

A number of transition metal ions with a wide distribution in biological systems, e.g., Cu2+, Co2+ and Zn2+, are shown to affect markedly the chemical properties of melanins formed by the tyrosinase-catalysed oxidation of dopa. Acid decarboxylation and permanganate degradation provide evidence that melanins prepared in the presence of metal ions contain a high content of carboxyl groups arising from the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DICA) into the pigment polymer. Naturally occurring melanins from cephalopod ink and B16 mouse melanoma were found to be much more similar to melanins prepared in the presence of metal ions than to standard melanins prepared in the absence of metal ions. These results suggest that the presence of carboxylated indole units in natural melanins is probably due to the intervention in the biochemical pathway of metal ions which, as recently shown, catalyse the formation of DICA versus 5,6-dihydroxyindole in the rearrangement of dopachrome.     

Complex inhibition of tyrosinase by thiol-composed Cu2+ chelators: a clue for designing whitening agents

The inhibition of tyrosinase has attracted considerable attention for potential medicinal and cosmetic applications, as well as in agriculture. This study investigated the inhibition effects of thiol-associated Cu(2+) chelators and deduced a strategy for designing and/or selecting tyrosinase inhibitors. Among the several compounds tested, dithioglycerine (DTGC) was selected for further experiments on the inhibition kinetics on tyrosinase. Different types of tyrosinases derived from mushroom and from the transient overexpression in HEK293 cells were tested individually. The results showed that DTGC significantly inhibited human tyrosinase in a complex manner (slope-parabolic mixed-type inhibition), which was comparable to mushroom tyrosinase. The affinity of DTGC affinity to human tyrosinase was evaluated by setting up a K(i slope) equation. The results suggest that a Cu(2+) chelator modified with thiol groups has potential as a whitening agent. In addition, a strategy for designing and/or selecting tyrosinase inhibitors that target the active enzyme site was also suggested.     

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.     

Oxidation Chemistry of Dopamine: Possible Insights into the Age-Dependent Loss of Dopaminergic Nigrostriatal Neurons

The oxidation chemistry of the chemical neurotransmitter dopamine (DA) has been investigated using electrochemical approaches. At physiological pH in aqueous solution DA is initially oxidized at a pyrolytic graphite electrode in a reversible 2e, 2H⁺ reaction to give DA-o-quinone (1). Deprotonation and intramolecular cyclization of 1 yields 5,6-dihydroxyin-doline (3) which is rapidly further oxidized (2e, 2H⁺) to 6-hydroxyindoline-6-one (4; dopaminochrome). Compound 4 then rearranges to 5,6-dihydroxyindole (5) which for the first time has been unequivocally identified as an oxidation product of DA. When preconcentrated on a preparative reversed-phase HPLC column 5 undergoes an unusual nonoxidative dimerization reaction to give 2-(5 ,6-dihydroxyindoline-3-yl)-5,6-dihydroxyindole (7). This 2,3′-linked indole-indoline dimer is extremely toxic when centrally administered to laboratory mice and evokes complex behavioral responses including convulsions, tremor, and hyperactivity. DA is known to undergo autoxidation in the cytoplasm of dopaminergic substantia nigra neurons to give neuromelanin polymer. Based on the results obtained in this study it is speculated that 5, a necessary intermediate in the latter reaction, is adsorbed on neuromelanin and reacts to form 7 which along with DA-o-quinone (1) and dopaminochrome (4) might be endotoxins that contribute to the age-dependent degeneration of dopaminergic SN neurons. In the presence of L-cysteine DA-o-quinone (1) reacts to give 5-S-cysteinyldopamine (8) not 6-S-cysteinyldopamine as has been suggested by other investigators.     

Homology models of four Agaricus bisporus tyrosinases

Partially purified tyrosinase from the white button mushroom Agaricus bisporus is available commercially and is a widely used experimental model for the study of tyrosinase. The structure of an H₂L₂ tetrameric form of the mushroom enzyme was recently determined by X-ray crystallography. In this structure the two H subunits originate from the PPO3 gene, and the two L subunits are formed by a protein of unknown function with a lectin-like fold. However, the X-ray structures and oligomeric states of the mushroom PPO1, PPO2, PPO4, and PPO5 gene products remain unknown. Commercial mushroom tyrosinase powder is a mixture containing several or all of these tyrosinases, so knowledge of their structures should provide insight regarding interpretation of experimental data generated using commercial preparations of the enzyme. The PPO3 structure (H-subunit) was used as a template to generate homology models for the structures of the other four tyrosinases, and the resulting structural models were evaluated. Due to the moderate to high percentage of sequence identity (∼37-76%) between PPO3 and the other four tyrosinases, the backbone conformations of the predicted structures are very similar to that of PPO3. The alpha carbons of the six copper-coordinating histidines in the active site are positioned properly in the predicted structures, but their side chains are not oriented optimally for copper binding in some cases. Thus, the models are likely to provide an accurate representation of the actual tertiary structures, but they may have limited use in studies involving docking of substrates or inhibitors in the active site. Comparison of the homology models to the structure of molluscan hemocyanin enabled a prediction of the orientation of the enzyme's C-terminal domain over the active site in the latent enzyme.     

Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases.

The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.