Indirect oxidation of 6-tetrahydrobiopterin by tyrosinase

6-Tetrahydrobiopterin is known to bind to an allosteric site of tyrosinase to directly inhibit the enzyme. However, simultaneous measurements of ultraviolet-visible absorption spectra and oxygen consumption led us to conclude that the inhibition was due to oxidation of 6-tetrahydrobiopterin by dopaquinone. Immediately after addition of 6-tetrahydrobiopterin, tyrosinase stopped producing dopachrome from either tyrosine or dopa. Duration of inhibition was proportional to the concentration of added 6-tetrahydrobiopterin and the enzyme activity was fully restored after the inhibition. Surprisingly, there was a rapid consumption of oxygen during the inhibition period. In addition, absorption spectra indicated that the only reaction that occurred during the inhibition was oxidation of 6-tetrahydrobiopterin to 7,8-dihydrobiopterin. In the absence of tyrosine or dopa, tyrosinase did not oxidize 6-tetrahydrobiopterin, suggesting that a reaction intermediate between dopa and dopachrome was a target for the inhibition. We propose a new mechanism in which dopa is oxidized to dopaquinone and the latter, instead of producing dopachrome, is reduced back to dopa by 6-tetrahydrobiopterin.     

Synthesis and tyrosinase inhibitory activity of novel N-hydroxybenzyl-N-nitrosohydroxylamines

Several novel N-substituted N-nitrosohydroxylamines were synthesized. They all inhibited mushroom tyrosinase, but the type of inhibition was different depending on the substituent. Some N-(mono- or dihydroxybenzyl)-N-nitrosohydroxylamines exhibited uncompetitive inhibition with respect to L-dopa. Among them, compound 6 was also a competitive inhibitor with respect to oxygen. This observation suggests that another interaction by the meta- or para-hydroxyl group might stabilize the binding of the inhibitor to the enzyme through the oxygen binding site.     

Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase

The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS-PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.     

Method for the determination of molar absorptivities of thiol adducts formed from diphenolic substrates of polyphenol oxidase

Metabolic thiols such as cysteine and glutathione are involved in the biosynthetic pathway of phaeomelanins. They attack the o-quinones generated by polyphenol oxidase in its action on mono and o-diphenols and thus generate adducts. Determination of the molar absorptivities of these adducts is useful for spectrophotometric studies of phaeomelanin biosynthesis, antibrowning reagents in plants, and polyphenol oxidase assay methods. For their calculation, a method based on the depletion of o-diphenol by the action of polyphenol oxidase in the presence of thiol has been proposed. However, the method is slow and presents certain problems, for which reason we propose a new and faster method based on the action of polyphenol oxidase on o-diphenols which are in excess with respect to oxygen. Under these assay conditions there is rapid enzymatic formation of o-quinones, which react stoichiometrically with a thiol giving rise to the corresponding thiol-diphenol adduct. The method has been successfully applied to adducts of cysteine and glutathione with several o-diphenolic substrates of polyphenol oxidase involved in phaeomelanin biosynthesis in skin, neurones, and plants.     

The Inhibitory Effect of Glabridin from Licorice Extracts on Melanogenesis and Inflammation

Glabridin is the main ingredient in hydrophobia fraction of licorice extract affecting on skins. In this study, we investigated inhibitory effects of glabridin on melanogenesis and inflammation using cultured B16 murine melanoma cells and guinea pig skins. The results indicated that glabridin inhibits tyrosinase activity of these cells at concentrations of 0.1 to 1.0 μg/ml and had no detectable effect on their DNA synthesis. Combined analysis of SDS-polyacrylamide gel electrophoresis and DOPA staining on the large granule fraction of these cells disclosed that glabridin decreased specifically the activities of Tl and T3 tyrosinase isozymes. It was also shown that UVB-induced pigmentation and erythema in the skins of guinea pigs were inhibited by topical applications of 0.5% glabridin. Anti-inflammatory effects of glabridin in vitro were also shown by its inhibition of superoxide anion productions and cyclooxygenase activities. These data indicated that glabridin is a unique compound possessing more than one function; not only the inhibition of melanogenesis but also the inhibition of inflammation in the skins. By replacing each of hydroxyl groups of glabridin with others, it was revealed that the inhibitory effect of 2′-O-ethyl glabridin was significantly stronger than that of 4′-O-ethyl-glabridin on melanin synthesis in cultured B16 cells at the concentration of 1.0 mg/inl. With replacement of both of two hydroxyl groups, the inhibitory effect was totally lost. Based on these data, we concluded that two hydroxyl groups of glabridin are important for the inhibition of melanin synthesis and that the hydroxyl group at the 4’ position of this compound is more closely related to melanin synthesis.     

Effect of Arbutin on Melanogenic Proteins in Human Melanocytes

The inhibitory effect of arbutin, a naturally occurring β-D-glucopyranoside derivative of hydroquinone, on melanogenesis was studied biochemically by using human melano-cytes in culture. Cells were cultured in the presence of different concentrations of arbutin. The maximum concentration of arbutin that was not inhibitory to growth of the cells was 100 ug/ml. At that concentration, melanin synthesis was inhibited significantly by ～20% after 5 days, compared with untreated cells. This phenotypic change was associated with the inhibition of tyrosinase and DHICA polymerase activities, and the degree of inhibition was dose dependent. No significant difference in DOPAchrome tautomerase (DT) activity was observed before or after arbutin treatment. Western blotting experiments revealed there were no changes in protein content or in molecular size of tyrosinase, TRP-1 or TRP-2, indicating that inhibition of tyrosinase activity by arbutin might be due to effects at the post-translational level.     

Calcium Plays a Complex Role in the Regulation of Melanogenesis in Murine B16 Melanoma Cells

To learn more of the role of calcium in the regulation of melanogenesis, we have used direct manipulation of medium calcium and pharmacological modulation of intracellular calcium to examine the consequences on unstimulated and cyclic AMP elevated tyrosinase activity and melanin synthesis and distribution in B16 melanoma cells. In unstimulated cells, calcium is clearly inhibitory to tyrosinase activity. However, in cells stimulated with cAMP-elevating agents the requirement for extracellular calcium was changed such that cells required a minimum of 0.4–0.6 mmol medium calcium for maximum tyrosinase response to these agents. Paradoxically, pharmacologically increasing intracellular calcium in cAMP-stimulated cells with ionophore inhibited tyrosinase activity, and the calcium-lowering agent TMB8 and the calcium channel blocker verapamil both stimulated tyrosinase activity. When melanin synthesis was measured in cAMP-stimulated cells, TMB8 was found to significantly increase the sensitivity and the maximum melanogenic response to α-MSH, suggesting the presence of at least one level of endogenous calcium inhibitory control operative in these cells. In addition, TMB8 changed the distribution of melanin between the cell and the medium such that, in the presence of α-MSH and TMB8, significantly more melanin was secreted into the medium. These data suggest that calcium is required for several steps in melanogenesis, having an apparently inhibitory effect on pre-tyrosinase activity in unstimulated cells, but also showing evidence of a positive role in cyclic AMP-stimulated tyrosinase activity, as well as a further possible inhibitory role in melanin movement or secretion.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.