千金子不同极性部位对酪氨酸酶活性的影响

测定千金子醇提物不同极性部位对酪氨酸酶活性的影响。以L-酪氨酸为底物,采用比色法测定千金子不同极性部位对酪氨酸酶的抑制率。得到了千金子醇提物的乙酸乙酯相、正丁醇相和水相的半数抑制率(IC50值)分别为0.150 mg/mL,0.813 mg/mL,7.570 mg/mL,并对乙酸乙酯相进一步分离得到七叶内酯,其IC50值0.103 mg/mL。结果表明千金子中起到酪氨酸酶抑制性的物质为七叶内酯,主要分布在乙酸乙酯相中。为千金子中酪氨酸酶抑制性物质的筛选提供科学依据。     

Kinetic characterization of the oxidation of esculetin by polyphenol oxidase and peroxidase

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.     

Kinetic Characterization of the Oxidation of Esculetin by Polyphenol Oxidase and Peroxidase

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.     

Phenylpropanoids, phenylalanine ammonia lyase and peroxidases in elicitor-challenged cassava (Manihot esculenta) suspension cells and leaves

BACKGROUND AND AIMS: Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. METHODS: Phenylalanine ammonia-lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. KEY RESULTS: A single and rapid (> or =2-3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four-fold in cells, 48 h post-elicitation. POD isoforms (2-7 isoforms, pI 3.1-8.8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post-elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3.6, present in all samples. Unidentified phenolics and possibly scopolin increased post-elicitation, but there was no enhancement of scopoletin, rutin or kaempferol-3-O-rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by > or =50 microg mL(-1) of ferulic acid and quercetin and > or =10 microg mL(-1) of scopoletin. CONCLUSIONS: Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and scopoletin by peroxidase and of esculetin by tyrosinase enhanced their fungitoxicity up to 20-fold.     

Product deactivation in recombinant Streptomyces

The production of tyrosinase by Streptomyces antibioticus (p1J7O2) was investigated as a model system for recombinant protein production by Streptomyces. Product deactivation was found to have a severe effect on the levels of tyrosinase obtained. Tyrosinase deactivation was detected during all phases of batch cultures, with higher specific deactivation rates observed during the stationary phase. The specific deactivation rate exhibited an Arrhenius dependence on temperature, with approximately a twofold increase in the deactivation rate between 25 degrees C and 30 degrees C. The effect of deactivation on the determination of tyrosinase production kinetics is discussed. A strategy was implemented to increase tyrosinase productivity by enriching the growth medium and reducing the culture temperature during the period of maximum tyrosinase production. This strategy resulted in a shorter culture time and a 2.5-fold increase in tyrosinase activity compared to a culture grown at 25 degrees C using a standard growth medium.     

Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures

The activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, is higher in Black skin melanocytes than in melanocytes derived from Caucasian skin. This variation in enzyme activity is not due to differences in tyrosinase abundance or tyrosinase gene activity, but, rather, is due to differences in the catalytic activity of preexisting tyrosinase. In melanocytes, tyrosinase is localized to the membrane of melanosomes and in Caucasian melanocytes the melanosome-bound enzyme is largely inactive. Conversely, in melanosomes of Black melanocytes, tyrosinase has high catalytic activity. Treatment of Caucasian melanocytes with the lysosomotropic compound ammonium chloride or with the ionophores nigericin and monensin results in a rapid and pronounced increase in tyrosinase activity. This increase occurs without any change in tyrosinase abundance, indicating that these compounds are increasing the catalytic activity of preexisting enzyme. Inhibition of the vacuolar proton pump V-ATPase by treatment of Caucasian melanocytes with bafilomycin also increases tyrosinase activity. In contrast to the 10-fold increase in tyrosinase observed in Caucasian melanocytes, neither ammonium chloride, monensin, nigericin, nor bafilomycin is able to increase the already high level of tyrosinase activity present in melanosomes of melanocytes derived from Black skin. Finally, staining of Caucasian melanocytes with the fluorescent weak base acridine orange shows that melanosomes of Caucasian, but not Black, melanocytes are acidic organelles. These data support a model for racial pigmentation that is based on differences in melanosome pH in Black and Caucasian skin types. The models suggests that melanosomes of Caucasian melanocytes are acidic, while those of Black individuals are more neutral. Since tyrosinase is inactive in an acid environment, the enzyme is largely inactive in Caucasian melanosomes but fully active in Black melanosomes.     

慢性应激通过减少毛囊黑色素细胞和酪氨酸酶活性导致雌性C57小鼠毛发颜色变浅

越来越多的证据表明压力可能会导致头发颜色发生变化,但其潜在机制尚不完全清楚。本研究采用雌性C57BL/6小鼠脚底电刺激结合束缚来建立慢性应激小鼠模型,并用比色法检测小鼠皮肤和B16F10黑色素瘤细胞中黑色素含量和酪氨酸酶活性;通过酶联免疫吸附实验(ELISA)测定小鼠皮肤中肿瘤坏死因子α(tumor necrosis factorα, TNF-α)、白细胞介素1β(interleukin-1β, IL-1β)和白细胞介素6 (interleukin-6, IL-6)含量;通过免疫荧光染色评估小鼠皮肤中核因子κB (nuclear factorκB, NFκB)/p65亚基的含量。结果显示:C57BL/6小鼠在慢性应激下由于皮肤中的毛囊黑色素细胞和酪氨酸酶活性降低,其毛皮颜色从暗色变为棕色。同时,慢性应激小鼠皮肤炎症反应增加,表现为皮肤中NFκB活性和TNF-α表达增加。在体外,TNF-α以剂量依赖性方式降低B16F10黑色素瘤细胞中黑色素生成和酪氨酸酶活性。以上结果表明,慢性应激通过降低雌性C57BL/6小鼠的毛囊黑色素细胞和酪氨酸酶活性来诱导皮毛颜色改变,而TNF-α可能在应激诱导的毛色改变中起重要作用。     

pH-dependent interconvertible forms of mushroom tyrosinase with different kinetic properties

The lag in cresolase activity and inhibition by excess tyrosine of mushroom tyrosinase which was observed when assayed at pH 6.8 was found to be absent when assayed at pH 5.0. The absence of lag and inhibition by excess tyrosine of tyrosinase at pH 5.0 were brought about only after the enzyme was kept at pH 5.0, at 0-4 degrees C, for 1.5 h. The enzyme kept at pH 5.0 for 1.5-3 h at 0-4 degrees C when brought back to pH 6.8, acquires lag and inhibition by excess tyrosine when its activity was measured at pH 6.8. The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin, and thus appear to be a general property of tyrosinase from diverse sources.     

Regulation of rat natural killing. II. Inhibition of cytolysis and activation by inhibitors of lipoxygenase: possible role of leukotrienes

Rat splenic natural killer (NK) cell activity against 51Cr-labeled YAC-1 or TMT-081 tumor cells can be augmented by culturing at 37 degrees C for 18 hr. Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism, NDGA, alpha-phenanthroline, quercetin, ETYA, BW755C, esculetin, and timegadine, inhibited this NK activation and also inhibited NK cytotoxicity when added directly to the NK assay. However, there was a partial loss of sensitivity of activated NK cells to suppression by NDGA, BW755C, and esculetin. Indomethacin failed to reverse the inhibition of NK activation caused by NDGA. However, LTB4 and LTC4 (0.01 microgram/ml) were able to reverse the inhibitory effect of NDGA on NK activation. Furthermore, spleen cells cultured for 18 hr synthesized detectable amount of LTC4 in their supernatants. NDGA inhibited the LTC4 synthesis in a dose-dependent manner. These data therefore suggest that leukotrienes are responsible for NK activation, and lipoxygenase activity is essential for NK cytolytic activity.     

A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37 degrees C but exits the ER and accumulates in endosomal structures in cells grown at 31 degrees C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37 degrees C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37 degrees C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.     

Effects of sugars on melanogenesis in cultured melanoma cells.

A permanent cell line C2M of mouse melanoma B16 was highly melanized in a modified Eagle's MEM supplemented with 10% calf serum, when the medium contained 1 mM galactose and 10 mM pyruvate instead of 5.5 mM glucose. The activity of the key anzyme for melanogenesis, tyrosinase (EC 1.14.18.1), of living cells cultured in the galactose-pyruvate medium was consistently 27 times higher than that of cells in normal MEM. This high level of tyrosinase activity was maintained in the stationary phase, in contrast to the activity of cells in normal medium, which decreased sharply in the stationary phase. It seems likely that tyrosinase activity is suppressed by the presence of glucose rather than stimulated by galactose. This modified medium should be useful obtaining a high level of tyrosinase activity in living cells in culture and in cell-free extracts.     

Antioxidant activity of hydroxy derivatives of coumarin

The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H2O2-tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (K(i), microM) and the inhibition degree (%). The K(i) values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 microM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity ofesculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K(i) value equal to 1.15 microM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.     

Regulation of Melanogenesis Induced by 5-Methoxypsoralen Without Ultraviolet Light in Murine Melanoma Cells

Melanogenesis in melanoma cells can be enhanced by psoralens in the absence of UV light. Melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase, DOPAchrome tautomerase (DCT), and tyrosinase-related protein-1 (TRP-1, gp75). To get more insight on the molecular mechanisms involved in psoralens-induced melanogenesis, we determined tyrosinase and DCT activities as well as mRNA and protein levels of tyrosinase, DCT, and TRP-1 in S91 mouse melanoma cells treated by 5-MOP. High concentration of 5-MOP (5 × 10^-5 M) induced a time-dependent increase of tyrosinase activity and melanin content, which was correlated to an increase of both mRNA and protein levels of tyrosinase. These results demonstrate that the 5-MOP stimulation of melanogenesis is related to increased tyrosinase synthesis. In addition, 5-MOP stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without any modification in the expression of the protein. We explored then the signalling pathways involved in 5-MOP-induced melanogenesis and, particularly, the role of cyclic AMP and protein kinase C (PKC). A small stimulation of cyclic AMP production was observed in presence of 5-MOP. Furthermore, 1-oleoyl-2-acetylglycerol (OAG), a PKC activator, potentiated the 5-MOP stimulation of tyrosinase activity, while calphostin, a specific PKC inhibitor, inhibited the 5-MOP induction of tyrosinase activity. Phorbol-myristate acetate (PMA), described as a strong activator of PKC, inhibited also the effect of 5-MOP when used at long term. Taken together, these results demonstrate that in murine melanoma cells 5-MOP stimulates melanogenesis by increasing activity and synthesis of tyrosinase. Tyrosinase and TRP-1 expression are coordinately regulated by 5-MOP Furthermore, a negative correlation between melanogenesis and DCT activity was observed under 5-MOP stimulation. At least, PKA and PKC systems appear to play an important role in the melanogenic effect of 5-MOP.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKCα had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKCα in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1. © 1996 Wiley-Liss, Inc.     

Regulation of tyrosinase in human melanocytes grown in culture

Tyrosinase, the enzyme that controls the synthesis of melanin, is a unique product of melanocytes. Normal and malignant human melanocytes grown in culture were used to study the factors that regulate the expression of tyrosinase. Immunoprecipitation experiments showed that newly synthesized tyrosinase appeared as a protein with an apparent molecular weight of 70,000 that was processed to a protein with an apparent molecular weight of 80,000. Neither tunicamycin nor 2-deoxy-D- glucose inhibited this conversion, suggesting that O-glycosylation is the major biochemical event in the posttranslational modification of tyrosinase. Agents that stimulated the proliferation of normal melanocytes also stimulated tyrosinase activity. Melanocytes with low levels of tyrosinase activity synthesized less tyrosinase, processed the enzyme more slowly, and degraded it more rapidly than melanocytes with high levels of tyrosinase activity. We conclude that tyrosinase activity in cultures of human melanocytes derived from different donors is determined predominantly by its abundance.     

Synthesis and degradation of tyrosinase in cultured melanoma cells

The tyrosinase (EC 1.14.18.1) activity of cultured B-16 mouse melanoma cells (C2M) in the stationary phase depends greatly on whether the culture medium contains glucose or galactose. The activity in medium containing galactose was about ten times that in medium containing glucose at pH 7.2. This difference in tyrosinase activity was concluded to be due to a shift of balance between synthesis and degradation of the enzyme. Experiments were conducted with stationary phase cultures in the presence of cycloheximide. The melanoma cells did not synthesize tyrosinase in medium containing glucose in the stationary phase. But when they were cultured under identical conditions, except that glucose was replaced by galactose, they continued to synthesize tyrosinase. The rate of synthesis in medium containing galactose at pH 6.3 was one third of that in the same medium at about pH 7, in which the increase in specific activity of tyrosinase per day was about 30 nmoles/mg cell protein per hr. The rate of degradation of the enzyme was practically the same in medium containing glucose as in medium containing galactose, and largely depended on the pH of the culture medium. At pH 6.3, the half-life was about one third of that at pH 7.2, where it was about 1.8 days. The degradation at acidic pH values was much reduced by ammonium salt and was strongly inhibited by the protease inhibitor, leupeptin.     

Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media

The enzymatic activity of mushroom tyrosinase was investigated using catechin as substrate in selected organic solvent media. The results showed that optimal tyrosinase activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in the organic solvent media of heptane, toluene, dichloromethane, and dichloroethane, respectively, and at a temperature between 25°C and 27.5°C. In addition, the kinetic studies showed that the K_m values were 5.38, 1.03, 2.52 and 4.03 mM, for the tyrosinase-catechin biocatalysis in the reaction media of heptane, toluene, dichloromethane, and dichloroethane, respectively, while the corresponding V_max values were 1.22×10⁻³, 0.33×10⁻³, 1.47×10⁻³ and 1.20×10⁻³ δA per μg protein per second, respectively. The use of acetone as co-solvent for the tyrosinase-catechin biocatalysis showed that acetone concentrations ranging from 5% to 30% (v/v) in the heptane reaction medium produced a decrease of 4.3% to 96.7% in tyrosinase activity. The results also indicated that the presence of 12.5% acetone in the reaction medium of dichloromethane, and 22.0% in those of toluene and dichloroethane produced a maximal increase of 42.6%, 92.1% and 71.8%, respectively, in tyrosinase activity. However, the overall findings indicated that additional increases in acetone concentration resulted in an inhibition of tyrosinase activity.     

Antioxidant activity of hydroxy derivatives of coumarin

The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H₂O₂-tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K _i, μM) and the inhibition degree (%). The K _i values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 μM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity of esculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K _i value equal to 1.15 μM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.