Browsing by Subject "Tyrosinase"

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    A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase
    (Wiley, 2010-04-13) Jiménez-Atiénzar, Mercedes; Cabanes Cos, Juana; Escribano Cebrián, Josefa; Gandía Herrero, Fernando; Pérez Gilabert, Manuela; García Carmona, Francisco; Departamento de Bioquímica y Biología Molecular "A"
    Introduction – Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some orna-mental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase fromthe chalcone THC (2′,4′,6′,4–tetrahydroxychalcone).Objective – To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kineticcharacteristics of THC oxidation by tyrosinase.Methodology – THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time.Results – According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o-hydroxylation of themonophenol THC to the o-diphenol (PHC, 2′,4′,6′,3,4 – pentahydroxychalcone), which is then oxidised to the correspondingo-quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactionsto produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curvesobtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined:Km = 0.12 mM , Vm = 13 mM /min, Vm/Km = 0.11/min.Conclusion – This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyro-sinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and repro-ducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone.
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    Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review
    (Elsevier, 2024-04-10) García Molina, Pablo; Saura Sanmartín, Adrián; Berná Cánovas, José; Muñoz Muñoz, Jose Luis; García Cánovas, Francisco; García Molina, Francisco; Rodríguez López, José Neptuno; Teruel Puche, José Antonio; Saura Sanmartín, Adrián; García Molina, Francisco; Química Orgánica
    Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of ICM 50 (monophenolase) and ICD 50(diphenolase). Addi tionally, the strength and type of inhibition can be deduced from these values. The data analysis from these ICD 50 values allows to obtain the values of Kapp I1 or Kapp I2 , or Kapp I1 and Kapp I3 from the values of ICM 50. In all cases, the values of the different Kapp I must satisfy their relationship with ICM 50 and ICD 50.
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    Genetics of pigment cells, lessons from the tyrosinase gene family
    (Murcia : F. Hernández, 2006) Murisier, F.; Beermann, Friedrich
    In mammals, the melanin pigment is produced in two cell types of distinct developmental origins. The melanocytes of the skin originate form the neural crest whereas the retinal pigment epithelium (RPE) of the eye originates from the optic cup. The genetic programs governing these two cell types are thus quite different but have evolved to allow the expression of pigment cell-specific genes such as the three members of the tyrosinase-related family. Tyrosinase, Tyrp1 and Dct promoters contain a motif termed E-box which is bound by the transcription factor Mitf. These E-boxes are also found in the promoters of the corresponding fish genes, thus highlighting the pivotal role of Mitf in pigment cell-specific gene regulation. Mitf, which displays cell type-specific isoforms, transactivates the promoters of the tyrosinase gene family in both pigment cell lineages. However, specific DNA motifs have been found in these promoters, and they correspond to binding sites for RPE-specific factors such as Otx2 or for melanocyte-specific factors such as Sox10 or Pax3. The regulation of pigment cell-specific expression is also controlled by genetic elements located outside of the promoter, such as the tyrosinase distal regulatory element located at -15 kb which acts as a melanocytespecific enhancer but also protects from spreading of condensed chromatin. Thus, by using the tyrosinase gene family as a model, it is possible to define the transcription factor networks that govern pigment production in either melanocytes or RPE.
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    Molecular docking studies of ortho-substituted phenols to tyrosinase helps discern if a molecule can be an enzyme substrate
    (MDPI, 2024-06-23) Garcia-Molina, Pablo; Garcia-Canovas, Francisco; Garcia-Molina, Francisco; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Tudela Serrano, José; Bioquímica y Biología Molecuar A
    Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring.
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    Molecular docking studies of ortho-substituted phenols to tyrosinase helps discern if a molecule can be an enzyme substrate
    (MDPI, 2024-06-23) García-Molina, Pablo; García-Cánovas, Francisco; García-Molina, Francisco; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Teruel Puche, José Antonio; Tudela Serrano, José; Bioquímica y Biología Molecular A
    Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring.
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    The melanogenic system of the liver pigmented macrophages of Rana esculenta L. - Tyrosinase activity
    (Murcia : F. Hernández, 2007) Gallone, A.; Sagliano, A.; Guida, G.; Ito, S.; Wakamatsu, K.; Capozzi, V.; Perna, G.; Zanna, P.; Cicero, R.
    The enzyme system responsible for Amphibian Kupffer Cell (KC) melanogenesis has not been entirely elucidated. This research demonstrates that the KC melanosomes of Rana esculenta L. possess a tyrosine-hydroxylase (TH) activity, showing that a tyrosinase is the enzyme involved in the melanogenesis. The TH reaction depends on catalytic Dopa as a cofactor and is not affected by catalase or H2O2, showing that it is catalysed by the tyrosinase and not by the peroxidase present in the melanosomes. The TH reaction is activated by Cu2+ ions but not by other tyrosinase activators such as limited proteolysis, protein ageing, and Sodium Dodecyl Sulphate (SDS). SDS inhibited the KC TH activity even below the critical micelle concentration. All these results suggest that the KC-tyrosinase differs in structure from other known tyrosinases. Using anti-KC-tyrosinase antobodies, we observed that the sites of the tyrosinase location within the cell are the same as those described in the melanocytes. In the immunoblots, the anti-KC-tyrosinase antibodies also recognised two protein bands, at the higher molecular weight ranges, in the protein electrophoretic pattern. Moreover, the tyrosinase activity was limited to the highest molecular weight band of about 260 kDa, suggesting that the enzyme activity could depend on a molecular aggregate. The melanin produced in the liver was found to be a 5,6-dihydroxyindole-rich eumelanin similar to the Sepia melanin.