Tyrosinase in the presumptive pigment cells of ascidian embryos: Tyrosine accessibility may initiate melanin synthesis

Tyrosinase activity appears in the presumptive pigment cells of ascidian embryos (Ciona intestinalis) several hours before the cells begin to synthesize melanin. These presumptive pigment cells develop into the otolith and ocellus pigment cells of the larval brain. Tyrosinase was identified by histochemical tests for tyrosine oxidase and dopa oxidase; both reactions were sensitive to tyrosinase inhibitors. Studies with puromycin suggested that tyrosinase was synthesized at the time it was first detected histochemically and that it was stable during the time interval before melanin synthesis. Supernumerary tyrosinase-containing cells were found adjacent to the presumptive pigment cells in three ascidian species examined (C. intestinalis, Styela partita, and Molgula manhattensis). Tyrosinase disappeared from the supernumerary pigment cells during larval development and these cells did not synthesize melanin.Tyrosinase in the presumptive and supernumerary pigment cells is apparently a functional enzyme which does not interact with substrate. External substrates ( -tyrosine and -dopa) did not react with enzyme in the living cells before the normal time of pigment synthesis, but gentle disruption of the cells (by freezing-and-thawing or osmotic shock) released active tyrosinase. Progessive enlargement of nonpigmented vesicles in the otolith cells of embryos exposed to phenylthiourea, an inhibitor of tyrosinase activity, suggested that tyrosinase vesicles actively accumulate tyrosine at the beginning of melanin synthesis. This tyrosine accumulation probably initiates melanin synthesis.     

Limitations of the quantitative cytochemical assay of catechol oxidase in melanoma cells

Summary The cytochemical quantification of catechol oxidase activity in fixed B16 melanoma cells was investigated using dopa as the substrate. Inhibitors showed that peroxidases do not significantly interfere. The kinetics of melanin formation were studied initially in solution with purified catechol oxidase. Two key parameters were identified: lag-time and the rate of melanin formation. The lag-time was taken as the time required by intermediates to reach a critical concentration at which the polymerization process starts and melanin production becomes measurable (at 640 nm). In solution, the lag-time decreases as the enzyme activity increases, particularly when the activity is very low. The rate at which melanin is formed by pure enzyme in solution is independent of dopa concentration when its activity is low but increases linearly with dopa concentration when the activity is comparatively high.In fixed melanoma cells, the lag-time decreases linearly with increases of dopa concentrations up to 20mm; at concentrations higher than this, the lag decreases more slowly. In contrast, the rate of melanin production is unaffected by changes in dopa concentration. The lag-times of different cells lines incubated at the same substrate concentration decrease as the enzyme activity of the cells increases. The rate of melanin production seems to be affected by factors other than catechol oxidase activity, such as the intracellular organization and distribution of the enzyme.     

The synthesis and activity of tyrosinase during development of the frog Rana pipiens

We have established by radioimmunoprecipitation that tyrosine-DOPA oxidase (TDO, tyrosinase) [EC 1.14.18.1] is first synthesized by frog embryos at the early neurula stage soon after embryonic induction of the neural plate by the underlying chordamesoderm. The DOPA moiety of the enzyme, at the time of its first appearance, is almost inactive enzymatically and can be activated by mild proteolysis (with trypsin). A very large increase in the amount of active DOPA oxidizing enzyme (without trypsinization) is observed at hatching (stage 21), and this is accompanied by melanin deposition in pigment cells. The tyrosine moiety of the enzyme is also partially inactive at the time of first synthesis, but the ratio of active to inactive enzyme remains approximately constant throughout early development. DOPA decarboxylase enzymatic activity is first detected at neurula stage, and this activity is accompanied by the first appearance of catechol amines.     

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O₂ consumption during B. bufo development.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Assays for mammalian tyrosinase: a comparative study

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.     

Repeated-batch fermentation using flocculent hybrid, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CHFY0321 for efficient production of bioethanol

A new tyrosinase was isolated from Aeromonas media strain WS and purified to homogeneity. The purified tyrosinase, termed TyrA, had a molecular mass of 58 kDa and an isoelectric point of 4.90. It exhibited optimal monophenol and diphenol oxidase activities under basic conditions (pH > 8.0). TyrA had a relatively higher affinity to diphenol substrate l-dihydroxyphenylalanine (l-dopa) than many other tyrosinases. EDTA or glutathione notably inhibited the enzymatic activities of TyrA, whereas Triton X-100 and SDS activated them. The full-length TyrA gene was cloned, and it encodes a 518 amino acid protein with little similarities to other reported tyrosinases. However, the purified recombinant TyrA expressed in Escherichia coli demonstrated tyrosinase activity. These results suggest that TyrA is the first reported distinct tyrosinase involved in melanin production in the genus Aeromonas.     

Activation of tyrosinase in mouse melanoma cell cultures

Summary Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37°C for a minimum of 8 h. Enzyme activity continued to increase for 48h at which time the maximal level of activation was observed. Activation did not occur at 4°C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation process increased the V _Max of tyrosinase 10-fold and lowered the K _M by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole. The “self-activation” response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells. This investigation was supported by Public Health Service grants CA41425 and CA30393 awarded by the National Cancer Institute, Bethesda, MD and by a research grant from the Proctor and Gamble Company.     

Down-regulation of melanin synthesis by a biphenyl derivative and its mechanism

Down-regulation of melanin synthesis is required for recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened several phenolic derivatives using B16 melanoma cells and found that a biphenyl derivative, 2,2'-dihydroxy-5,5'-dipropyl-biphenyl (DDB), down-regulated melanin synthesis effectively. Although DDB has a phenol structure, it did not inhibit tyrosinase in vitro, thus we examined its mechanism in detail. Western blotting revealed that the amount of tyrosinase was decreased by DDB, and pulse-chase labeling and immunoprecipitation analysis showed a decrease of mature tyrosinase and acceleration of tyrosinase degradation in its presence. These results suggest that DDB down-regulates melanin synthesis by inhibiting the maturation of tyrosinase, leading to acceleration of tyrosinase degradation.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

金线莲抑制斑马鱼黑色素形成的活性组分筛选及机理研究

为探索金线莲中对黑色素形成具有抑制效果的活性组分,本研究对金线莲进行分离、提取,获得总提组、醇沉组与醇提组,利用斑马鱼筛选金线莲具有美白作用的活性组分。将受精后0.75 h的斑马鱼胚胎分别暴露于不同浓度的金线莲总提组、醇沉组、醇提组,72 h时观察结果表明,金线莲醇提物能有效抑制斑马鱼胚胎黑色素和黄色素沉着,浓度越高抑制效果越明显,且不影响胚胎生长发育。进一步采用半定量PCR和整胚原位杂交技术定量和定性地检测黑色素形成相关基因mRNA表达,结果表明金线莲醇提物可以有效降低silv、tyr和tyrp1a等黑色素合成相关基因的转录水平,且具有浓度依赖关系。通过检测酪氨酸酶活性显示,加入醇提物的实验组其酪氨酸酶活性随着处理浓度升高而逐渐降低。此外,在黑色素已经大量形成的情况下,金线莲醇提物仍可通过下调黑色素合成相关基因的mRNA表达及酪氨酸酶活性来抑制黑色素的形成,并且这种抑制效果可在金线莲醇提物撤除后得到恢复。上述实验结果表明,金线莲醇提物能显著抑制斑马鱼黑色素的形成,本文为金线莲在美白产品领域的开发和应用方面提供了有力支撑。     

Down‐Regulation of Melanin Synthesis by a Biphenyl Derivative and Its Mechanism

Down‐regulation of melanin synthesis is required for recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened several phenolic derivatives using B16 melanoma cells and found that a biphenyl derivative, 2,2′‐dihydroxy‐5,5′‐dipropyl‐biphenyl (DDB), down‐regulated melanin synthesis effectively. Although DDB has a phenol structure, it did not inhibit tyrosinase in vitro, thus we examined its mechanism in detail. Western blotting revealed that the amount of tyrosinase was decreased by DDB, and pulse‐chase labeling and immunoprecipitation analysis showed a decrease of mature tyrosinase and acceleration of tyrosinase degradation in its presence. These results suggest that DDB down‐regulates melanin synthesis by inhibiting the maturation of tyrosinase, leading to acceleration of tyrosinase degradation.     

Tyrosinase involved in betalain biosynthesis of higher plants

A tyrosine-hydroxylating enzyme was partially purified from betacyanin-producing callus cultures of Portulaca grandiflora Hook. by using hydroxyapatite chromatography and gel filtration. It was characterized as a tyrosinase (EC 1.14.18.1 and EC 1.10.3.1) by inhibition experiments with copper-chelating agents and detection of concomitant o-diphenol oxidase activity. The tyrosinase catalysed both the formation of L-(3,4-dihydroxyphenyl)-alanine (Dopa) and cyclo-Dopa which are the pivotal precursors in betalain biosynthesis. The hydroxylating activity with a pH optimum of 5.7 was specific for L-tyrosine and exhibited reaction velocities with L-tyrosine and D-tyrosine in a ratio of 1:0.2. Other monophenolic substrates tested were not accepted. The enzyme appeared to be a monomer with an apparent molecular mass of ca. 53 kDa as estimated by gel filtration and SDS-PAGE. Some other betalain-producing plants and cell cultures were screened for tyrosinase activity; however, activities could only be detected in red callus cultures and plants of P. grandiflora as well as in plants, hairy roots and cell cultures of Beta vulgaris L. subsp. vulgaris (Garden Beet Group), showing a clear correlation between enzyme activity and betacyanin content in young B. vulgaris plants. We propose that this tyrosinase is specifically involved in the betalain biosynthesis of higher plants. Received: 14 July 1998 / Accepted: 23 October 1998     

Redefining the skin's pigmentary system with a novel tyrosinase assay

In mammalian skin, melanin is produced by melanocytes and transferred to epithelial cells, with the epithelial cells thought to receive pigment only and not generate it. Melanin formation requires the enzyme tyrosinase, which catalyzes multiple reactions in the melanin biosynthetic pathway. Here, we reassess cutaneous melanogenesis using tyramide-based tyrosinase assay (TTA), a simple test for tyrosinase activity in situ. In the TTA procedure, tyrosinase reacts with biotinyl tyramide, causing the substrate to deposit near the enzyme. These biotinylated deposits are then visualized with streptavidin conjugated to a fluorescent dye. In the skin and eye, TTA was highly specific for tyrosinase and served as a sensitive indicator of pigment cell distribution and status. In clinical skin samples, the assay detected pigment cell defects, such as melanocytic nevi and vitiligo, providing confirmation of medical diagnoses. In murine skin, TTA identified a new tyrosinase-positive cell type--the medullary cells of the hair--providing the first example of cutaneous epithelial cells with a melanogenic activity. Presumably, the epithelial tyrosinase originates in melanocytes and is acquired by medullary cells during pigment transfer. As tyrosinase by itself can generate pigment from tyrosine, it is likely that medullary cells produce melanin de novo. Thus, we propose that melanocytes convert medullary cells into pigment cells by transfer of the melanogenic apparatus, an unusual mechanism of differentiation that expands the skin's pigmentary system.     

Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors

Melanogenesis is a process to synthesize melanin, which is a primary responsible for the pigmentation of human skin, eye and hair. Although numerous enzymatic catalyzed and chemical reactions are involved in melanogenesis process, the enzymes such as tyrosinase and tyrosinase-related protein-1 (TRP-1) and TRP-2 played a major role in melanin synthesis. Specifically, tyrosinase is a key enzyme, which catalyzes a rate-limiting step of the melanin synthesis, and the downregulation of tyrosinase is the most prominent approach for the development of melanogenesis inhibitors. Therefore, numerous inhibitors that target tyrosinase have been developed in recent years. The review focuses on the recent discovery of tyrosinase inhibitors that are directly involved in the inhibition of tyrosinase catalytic activity and functionality from all sources, including laboratory synthetic methods, natural products, virtual screening and structure-based molecular docking studies.     

Tyrosinase activities of cell-free extracts and living cells of cultured melanoma cells

Melanogenesis in the course of monolayer culture of a stably melanotic clonal line C₂M, derived from a mouse melanoma B 16, was investigated. Tyrosinase activity per cell of cell-free extracts was highest when the extract was prepared from cells in the mid-exponential phase of growth, when it was more than 6 times the activity of that prepared from a fully grown culture or a culture in the very early phase. On the other hand, the enzyme activity per cell of living cells in culture was highest in the early phase of culture and decreased rapidly to a level of less than one tenth of the maximum activity, in the stationary phase.The upper limit of population density of cultured melanoma cells permissive for melanin synthesis (2 to 3 × 10⁵ cells/cm²) was much higher than that of normal (nonneoplastic) melanocytes, which had been reported to produce melanin only under conditions of clonal growth.The relative efficiency of tyrosinase activity in situ, expressed by the ratio of tyrosinase activity in culture to that of cell-free extract, decreased rapidly in the exponential phase of growth. This decrease correlates to the cell density in the culture, and little if at all to the division rate, and suggests a suppressing mechanism of melanin synthesis working at the enzyme level.