Citrate activates tyrosinase from B-16 murine melanoma and human skin

Citrate stimulates cresolase activity of tyrosinase from B-16 murine melanoma and human skin. Maximal stimulation by citrate was obtained at 2 mM, and stimulation was decreased at higher concentrations. Citrate stimulates tyrosinase not only from mammalian sources but also from mushroom. The stimulation was not due to reversal of inhibition of enzyme activity by excess tyrosine. On rapid decrease in pH of the enzyme solution from 6.8 to 5.0-5.2, the enzyme is no longer inhibited by excess tyrosine even when its activity was assayed at pH 6.8. Citrate also stimulates this form of enzyme. However, the stimulation is more at acidic pH than at pH 6.8. At higher concentrations of citrate the stimulatory effect decreases at both pH 5.0 and pH 6.8. Inhibition of this enzyme occurs at higher concentrations (22 mM) at pH 6.8. The physiological role of stimulation of cresolase activity of tyrosinase by citrate is yet to be unravelled.     

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.     

The TYRP1‐mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains

Tyrosinases are melanocyte‐specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra‐melanosomal domains of tyrosinase, TYRtr (19–469), and tyrosinase‐related protein 1, TYRP1tr (25–472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero‐oligomeric complexes to maintain the protective function.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

驱虫斑鸠菊对B-16黑素瘤细胞酪氨酸酶活性及黑素生成影响

目的 :探讨驱虫斑鸠菊体外对酪氨酸酶活性影响 ,以及对小鼠B - 16黑素瘤细胞株细胞增殖、黑素合成以及细胞内酪氨酸酶的作用。方法 :利用四甲基偶氮唑蓝 (MTT)比色法测定药物对细胞增殖的影响 ;采用酶学方法研究药物对酪氨酸酶活性的影响 ;470nm比色法测定黑素含量。结果驱虫斑鸠菊体外可激活酪氨酸酶活性 ,增强B - 16鼠黑素瘤细胞增殖 ,提高酪氨酸酶和黑色素合成能力 ;对整体动物黑素细胞具有促进合成和分泌作用。结论在白癜风的治疗中 ,驱虫斑鸠菊可增强酪氨酸酶活性 ,进而促进黑素合成     

Oculocutaneous albinism type 1: link between mutations,tyrosinase conformational stability,and enzymatic activity

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site‐directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism.     

Purification and some properties of tyrosinase from aspergillus flavipes 56003

A method for isolation and purification of tyrosinase from the fungus Aspergillus flavipes 56003 was developed. The method includes extraction with water, concentration on DEAE-cellulose, gel-filtration on Acrylex P-150, and ion-exchange chromatography on DEAE-Toyopearl 650M. The tyrosinase was purified to apparent homogeneity according polyacrylamide gel electrophoresis and ultracentrifugation. The tyrosinase is a 130-kD protein with pI 4.6. It contains two copper atoms. The Km and Vmax for tyrosine hydroxylation are 0.3 mM and 1300 &mgr;moles/min per mg at pH 6.8, and for dehydrogenation of 3,4-dihydroxyphenylalanine (DOPA) they are 5 mM and 16000 &mgr;moles/min per mg, respectively. Hydroxylation of monophenols has a characteristic lag period. The rate of tyrosine and DOPA oxidation is maximal at pH 6.0-6.8. The half-life of the enzyme at 50 degrees C is 40 min. The hydroxylase activity of the tyrosinase is more stable at neutral pH, whereas the dehydrogenase activity is more stable at acidic pH (4.0). The absorption spectrum of the enzyme has a maximum at 290 mn and a shoulder in the 320-400-nm region.     

In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30°C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K_i) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K_i value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30°C at both pHs. Moreover, the effect of temperature on K_i value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

Tyrosinase exhibits lag at pH 6.8 under steady state concentrations of tyrosine and 3,4-dihydroxy phenyl alanine in melanoma tissue.

Tyrosine to dopa ratio determines the extent of lag in cresolase activity of tyrosinase when assayed at pH 6.8. The levels of tyrosine and dopa in B-16 murine melanoma tissue were found to be 213 and 13 mmoles/g fresh wt of tissue respectively. Cresolase activity of tyrosinase, when assayed at the above steady state levels of tyrosine and dopa at pH 6.8, exhibited a lag of 5-15 min depending on the amount of enzyme used in the assay mixture and the initial enzyme activity was zero. Under in vivo conditions, the enzyme with zero initial activity can not be active and therefore a far reaching conclusion is that tyrosine to dopa ratio may not regulate the enzyme activity, unlike under in vitro conditions. Possible modes of the regulation of tyrosinase under in vivo conditions are discussed.     

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.     

Comparison of TRPs From Murine and Human Malignant Melanocytes

Most of our knowledge of the mammalian tyrosinase related protein (TRP) activities is derived from studies using murine melanoma models, such as B16 or Cloudman S-91 melanocytes. Owing to the high degree of homology between the murine and human enzymes, it has been assumed that their kinetic behavior could be similar. However, the protein sequences at the metal binding sites of the murine and human enzymes show some differences of possible functional relevance. These differences are more significant in the metal-A site than in the metal-B site. By using three human melanoma cell lines (HBL, SCL, and BEU), we have studied the catalytic abilities of the human melanogenic enzymes in comparison to those obtained for the counterpart murine enzymes isolated from B16 melanoma. We have found that TRP2 extracted from all cell lines show dopachrome tautomerase activity, although the activity levels in human malignant melanocytes are much lower than in mouse cells. Reconstitution experiments of the human enzyme indicate that TRP2 has Zn at its metal binding-sites. Although mouse tyrosinase does not show DHICA oxidase activity, and this step of the melanogenesis pathway is specifically catalyzed by mouse TRP1, the human enzyme seems to recognize carboxylated indoles. Thus, human tyrosinase could display some residual DHICA oxidase activity, and the function of human TRP1 could differ from that of the murine protein. Attempts to clarify the nature of the metal cofactor in TRP1 were unsuccessful. The enzyme contains mostly Fe and Cu, but the reconstitution of the enzymatic activity from the apoprotein with these ions was not possible.     

Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.     

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.     

盐生植物盐地碱蓬酪氨酸酶的酶学特性

酪氨酸酶是植物甜菜素生物合成的限速酶,但是,其酶学特性尚不了解。以黑暗培养3d的盐地碱蓬幼苗为材料,采用NaF抽提、饱和硫酸铵沉淀法提取盐地碱蓬中的酪氨酸酶,以研究其酶学特性。结果表明,酪氨酸酶氧化活性的最适温度为35℃,最适pH值为6.6,最适条件下Km=1.09mmol·L-1,Vmax=71.43μmol·g-1（FW）·min-1;酪氨酸酶羟化活性的最适温度为40℃,最适pH值为6.6,最适条件下Km=3.16mmol·L-1,Vmax=0.645μmol·g-1（FW）·min-1。Na2S2O3是盐地碱蓬酪氨酸酶的强效抑制剂,0.05mol·L-1Na2S2O3几乎完全抑制酪氨酸酶氧化及羟化活性。而0.01mmol·L-1的Cu2＋可以显著激活酪氨酸酶的氧化及羟化活性,分别为对照的126%和128.2%。这些结果表明盐地碱蓬中酪氨酸酶的羟化活性是影响甜菜红素合成速率的关键,也为深入研究盐地碱蓬酪氨酸酶在甜菜素合成中的作用及其与环境之间的关系奠定了基础。     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations

Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, -dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.     

Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications

AIMS: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. METHODS AND RESULTS: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8-10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45.4 and 163.6 U g(-1) protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35-38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4.5-5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6-7, in a large temperature range (30-70 degrees C), and was stable below 60 degrees C. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. CONCLUSIONS: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg(-1) protein for monophenolase and diphenolase respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking.