Melanin Formation Research Articles

Purification and characterization of melanogenic enzyme tyrosinase from button mushroom.

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it's taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

A Ric8/synembryn homolog promotes Gpa1 and Gpa2 activation to respectively regulate cyclic AMP and pheromone signaling in Cryptococcus neoformans.

The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1(Q284L) allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1(Q284L) negatively affected its interaction with Ric8, whereas the activated Gpa2(Q203L) allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans.

Inducing zone lines and melanin formation for decorative purposes on North American western wood species, with emphasis on conifers

Twenty-one wood species native to the Pacific Northwest region of the United States, including conifers, were tested to determine if any of the commonly utilised melanin producing spalting fungi could generate sufficient spalting coverage to compete within the decorative woods market. Zone lines were readily produced by Xylaria polymorpha on Douglas fir, redwood, myrtle, and cottonwood. Blue stain from Scytalidium lignicola was prolific on myrtle and ash. These results indicate that a number of Pacific Northwest native wood species are suitable for controlled spalting, including two conifers. The prevalence of spalting on these wood species should lead to their increased value, and allow the Pacific Northwest to compete within the spalted woods market.

Microscopic Investigation on Fungal Pigment Formation and its Morphology in Wood Substrates

Melanin formation and assembly by fungi has largely been investigated mainly for its importance in pathogenesis, as well as to establish the functions and biosynthetic pathways of melanin formed during the process of successional wood decay. It is known that melanin formation varies based on fungal species, especially melanin produced by ascomycetes versus basidiomycetes, and that the mechanisms of melanin production by basidiomycetes are more complex and thus far not entirely elucidated. This study compares in vivo melanin formation by Oxyporus populinus in sugar maple and Fomes fomentarius in birch, and in vitro pigmentation by Trametes versicolor, Xylaria polymorpha and Inonotus hispidus in sugar maple and beech, with and without the influence of the melanin precursor, catechol. The results of this research indicate a bi- or multi-modal activity of melanin production and assembly by wood decay fungi, and identify possible variations in melanin formation mechanisms as influenced by fungal and wood species.

LYSOZYME AND DEFENSE PEPTIDES AS SUPPRESSORS OF PHENOLOXIDASE ACTIVITY IN Galleria mellonella

The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response.

A NOVEL TYPE OF HEMOCYTES LOCALIZING MELANIZATION WITH HIGH‐SPREADING BEHAVIOR IN MYTHIMNA SEPARATA

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.

Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.

Cryptococcus neoformans and Cryptococcus gattii are the two etiologic agents of cryptococcosis. They belong to the phylum Basidiomycota and can be readily distinguished from other pathogenic yeasts such as Candida by the presence of a polysaccharide capsule, formation of melanin, and urease activity, which all function as virulence determinants. Infection proceeds via inhalation and subsequent dissemination to the central nervous system to cause meningoencephalitis. The most common risk for cryptococcosis caused by C. neoformans is AIDS, whereas infections caused by C. gattii are more often reported in immunocompetent patients with undefined risk than in the immunocompromised. There have been many chapters, reviews, and books written on C. neoformans. The topics we focus on in this article include species description, pathogenesis, life cycle, capsule, and stress response, which serve to highlight the specializations in virulence that have occurred in this unique encapsulated melanin-forming yeast that causes global deaths estimated at more than 600,000 annually.

천궁(川芎)의 활용(活用)을 위한 『동의보감(東醫寶鑑)』 처방연구(處方硏究)

Objectives : Until now the study of Cnidii Rhizoma, hemorrhage, brain waves, such as ischemic brain injury, analgesic, effect overcome of the stress from pregnancy melanin formation and inhibiting effects skin whitening have been published regarding this article. Cnidii Rhizoma demonstrates its different abilities depending on the characteristics. This paper reported that effect of Cnidii Rhizoma in Dongeuibogam blended prescriptions as main medicine. In addition, by analyzing data, we studied about utilizing of Cnidii Rhizoma. Methods : Cnidii Rhizoma in Dongeuibogam Prescriptions as the main ingredient was built with database of 202 prescriptions. Thus analyzed data was summarized in detail.(Table-1) If there is no difference in the title of the prescription but in other case the configuration information is different, formulations 1 and 2 were divided by the table. Results : The following results were reached through investigations on the prescriptions usikng Cnidii Rhizoma as a key component. 1. Prescriptions taking Cnidii Rhizoma as a monarch drug are utilized for 40 therapeutic purposes. In particular, 12.3% of prescriptions appear in the chapter of head, and 10.8% of those appear in the chapter of women, and 9.4% of eye, 8.9% of child, 6.4% of wind disease respectively. 2. Prescriptions utilizing Cnidii Rhizoma as the main ingredient are used in the treatment of headache, dizziness and pregnancy hemorrhage fetal movement, premature birth and they are also used for treating 131 different types of disease. 3. The dosage of Cnidii Rhizoma in formulas is from 2pun(about 0.75g) to 5don(nearly 18.75g), however 1don(nearly 3.75g) has been taken the most for clinical application. 4. We find out that according to herbs or prescriptions, Cnidii Rhizoma has a variety of functions such as ascending & descending of energy. Samultang is the most useful base prescription which used the Cnidii Rhizoma as the main component. Conclusion : These results suggest that, Cnidii Rhizoma once-amount use (don nearly 3.75g) 4g in head, gynecology, ophthalmology, pediatrics and paralysis disease associated with oriental medicine resource development can be considered to be widely used These results suggest that Cnidii Rhizoma was used most with 1 don(4g) and can be widely used for the resource development to the disease such as brain, gynecology, ophthalmologhy, pediatrics and wind-associated symptoms.

Isolation, Characterization and Selection of Avermectin-Producing Streptomyces avermitilis Strains From Soil Samples.

Background:Streptomyces avermitilis, belonging to Actinomycetes, is specialized for production of avermectin, used as an anthelmintic and insecticidal agent. It is mostly found in soil and its isolation is very crucial for medically important avermectin production.Objectives:In the present study, 10 bacterial isolates lacking antimicrobial activities were isolated from the soil samples collected from different areas of Lahore, Pakistan.Materials and Methods:Three distinctive localities of Lahore were opted for soil assortment to isolate S. avermitilis. About 50 isolates of Streptomyces species were attained through selective prescreening procedures. All of these isolates were studied for production of the secondary metabolite, avermectin. Different test like soluble pigment color and melanin formation were used for identification. Biochemical characterizations of those isolates closely resembling the control in morphological characteristics, soluble pigment color and melanin formation tests were performed.Results:The 10 selected isolates were identified as the avermectin-producing strain by fermentation and characterized on ISP2 medium for aerial and reverse side mycelia color, soluble pigment color and melanin formation, in comparison with S. avermitilis DSM 41445. The best avermectin-producing isolate S1-C (10.15 mg/L) showed similar result as S. avermitilis DSM 41445, when subjected for culture characteristics analysis in different media along with biochemical characterization.Conclusions:From the results, it was concluded that agricultural lands around Pakistan Council of Scientific and Industrial Research (PCSIR) Campus Lahore were rich sources of industrially important Streptomyces, especially S. avermitilis.

Tyrosinase enzyme: 1. An overview on a pharmacological target.

The tyrosinase enzyme (EC 1.14.18.1) is an oxidoreductase inside the general enzyme classification and is involved in the oxidation and reduction process in the epidermis. These chemical reactions that the enzyme catalyzes are of principal importance in the melanogenesis process. This process of melanogenesis is related to the melanin formation, a heteropolymer of indolic nature that provides the different tonalities in the skin and helps to the protection from the ultraviolet radiation. However, a pigment overproduction, come up by the action of the tyrosinase, can cause different disorders in the skin related to the hyperpigmentation. Several studies mainly focused on the characteristics of the enzyme have been reported. In this work, an approximation to general aspects related to this enzyme is made. Besides, it is treated the researches that have been published in the part of the biochemical anatomy dealing with diseases associated with this protein (melanogenesis), its active place and its physiological states, the molecular mechanism, the methods carried out to detect the inhibitory activity, and the used substrates.

강활과 벌사상자 추출물의 피부 미백효과

본 연구의 목적은 강활과 벌사상자 추출물에 의한 피부 멜라닌 형성 저해와 안정성과 자극성 시험을 통하여 기능성 화장품의 사용 물질 가능성을 평가하고자 하였다. 본 실험에 사용된 추출물은 White-<TEX>$AK^{TM}$</TEX>이고 INCI 명칭은 Osthole이다. White-<TEX>$AK^{TM}$</TEX>의 주성분은 쿠마린이고 EC50 값은 마우스 멜라노마 B16 세포 시험에서 2.7ppm이였다. White-<TEX>$AK^{TM}$</TEX>는 알부틴보다 100배 낮은 농도에서 저해 효과를 보였다. 미백 효과의 주 기작은 티로시나제-관련 유전자 발현을 저해하는 것으로 생각되었다. White-<TEX>$AK^{TM}$</TEX> 5% 용액을 함유한 기본적인 에센스 처방을 피부에 적용 시 피부자극을 경감시켰다. White-<TEX>$AK^{TM}$</TEX>은 불투명한 용기에서 4주간 자외선 하에서 100%의 회수율을 보였고 <TEX>$50^{\circ}C$</TEX>에서 4주간 97%의 회수율을 보였다. 따라서 피부 미백효과와 자극성 경감효과가 있고 자외선 조건하에서 안정한 White-<TEX>$AK^{TM}$</TEX>은 화장품 처방에서 기능성 원료로 사용될 수 있을 것으로 사료된다. The objective of this study is to estimate the inhibition of skin melanin formation by extract of Angelica koreana and Cnidium monnieri and the possibility of functional cosmetic materials through anti-irritation and stability test. The extract used in this experiment is White-<TEX>$AK^{TM}$</TEX> and the INCI name is Osthole. The main component of White-<TEX>$AK^{TM}$</TEX> was identified as coumarin and EC50 value was 2.7ppm by mouse melanoma B 16 cell test. White-<TEX>$AK^{TM}$</TEX> showed inhibitory effects 100 times lower concentration than arbutin. The main mechanism for skin whitening effect thought to be inhibition of tyrosinase-related gene expression. The basic essence formulation of White-<TEX>$AK^{TM}$</TEX> 5% solution applied to the skin showed the effect of relieving skin irritation. White-<TEX>$AK^{TM}$</TEX> in an opaque container, under UV conditions for 4 weeks, and showed close to 100% recovery and 97% recovery under <TEX>$50^{\circ}C$</TEX> for 4 weeks. Therefore, it is thought that White-<TEX>$AK^{TM}$</TEX> which helps skin whitening, relieving skin irritation and stable in UV condition is able to be used as the functional component in the cosmetic formulation.

Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta.

Anti-melanogenic effects of δ-tocotrienol are associated with tyrosinase-related proteins and MAPK signaling pathway in B16 melanoma cells

Tocotrienols are known to possess potent antioxidant, anticancer, and cholesterol lowering activities. Being able to rapidly penetrate the skin, these vitamin E isoforms have been explored for potential treatment against melanoma. This study aimed to elucidate the mechanism involved in the anti-melanogenic effects of δ-tocotrienol (δT3) in B16 melanoma cells. Results showed that at 20μM of δT3 significantly inhibited melanin formation and ROS generation. Treatment with δT3 also effectively suppressed the expression of melanogenesis-related proteins, including MC1R, MITF, TYRP-1, and TYRP-2. More importantly, we observed that the mitogen-activated protein kinase (MAPK) pathway was involved in mediating δT3's inhibitory effect against melanin production. Specifically, δT3 treatment markedly induced the activation of extracellular signal-regulated kinases (ERK). The use of ERK activation inhibitor (PD98059) abrogated the δT3-mediated downregulation expression melanogenesis-related proteins and restored melanin production. Furthermore, siRNA targeting ERK effectively blocked the δT3-induced repression of tyrosinase and TYRP-1 expression. These results suggest that δT3's inhibitory effect against melanogenesis is mediated by the activation of ERK signaling, thereby resulting in downstream repression of melanogenesis-related proteins and the subsequent melanin production. These data provide insight to δT3's effect and the targeting of ERK signaling for treatment against melanogenesis.

Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol

2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells.

Inhibitory effects of novel synthetic methimazole derivatives on mushroom tyrosinase and melanogenesis

In this study, we synthesized 4 methimazole (2-mercapto-1-methylimidazole, MMI) derivatives. The kinetics of inhibition on mushroom tyrosinase by methimazole and its derivatives were investigated. The results indicated that tert-butyl 3-methyl-2-sulfanylidene-2,3-dihydro-1H-imidazole-1-carboxylate (compound 3; 3), 2-mercaptoimidazole (MI; compound 1; 1) and MMI (compound 2; 2) significantly inhibited tyrosinase activity in a dose-dependent manner, exhibiting an IC50 value of 1.50mM, 4.11mM, and 1.43mM. However, compound 4 (4), compound 5 (5), and compound 6 (6) exerted no inhibitory effect on mushroom tyrosinase activity. Kinetic analysis indicated that 3 was a noncompetitive tyrosinase inhibitor, whereas both 1 and 2 were exhibited as mixed-type tyrosinase inhibitors. Furthermore, 3 exerted a potent inhibitory effect on intracellular melanin formation in the B16/F10 murine melanoma cells and did not cause cytotoxicity, as 1 and 2 did.

A new insight into mushroom tyrosinase inhibitors: docking, pharmacophore-based virtual screening, and molecular modeling studies

Tyrosinase, a widely spread enzyme in micro-organisms, animals, and plants, participates in two rate-limiting steps in melanin formation pathway which is responsible for skin protection against UV lights’ harm whose functional deficiency result in serious dermatological diseases. This enzyme seems to be responsible for neuromelanin formation in human brain as well. In plants, the enzyme leads the browning pathway which is commonly observed in injured tissues that is economically very unfavorable. Among different types of tyrosinase, mushroom tyrosinase has the highest homology with the mammalian tyrosinase and the only commercial tyrosinase available. In this study, ligand-based pharmacophore drug discovery method was applied to rapidly identify mushroom tyrosinase enzyme inhibitors using virtual screening. The model pharmacophore of essential interactions was developed and refined studying already experimentally discovered potent inhibitors employing Docking analysis methodology. After pharmacophore virtual screening and binding modes prediction, 14 compounds from ZINC database were identified as potent inhibitors of mushroom tyrosinase which were classified into five groups according to their chemical structures. The inhibition behavior of the discovered compounds was further studied through Classical Molecular Dynamic Simulations and the conformational changes induced by the presence of the studied ligands were discussed and compared to those of the substrate, tyrosine. According to the obtained results, five novel leads are introduced to be further optimized or directly used as potent inhibitors of mushroom tyrosinase.

Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.

IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders.

High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase

Tyrosinases catalyze two initial reaction steps in the formation of melanin. Purification of tyrosinases had always been a process accompanied with various problems caused by enzymatic browning processes. Here, an approach is presented for the purification of the latent enzyme from mushrooms which averts and removes interfering compounds (e.g. polyphenols) in advance to the extraction process. The described method is supposed being well suitable as a general protein purification protocol from natural sources like fungi and plants.The purified enzyme was investigated in detail by means of mass spectrometry: its intact protein mass was determined as 64,247.3Da and it was identified as number four of in total six isoforms (PPO1–6) by means of sequence analysis. Some PTMs, strain specific sequence disparities and several cleavage sites including the one causing enzyme-activation (Ser383) were determined, thus, providing insights on the maturation process of this latent tyrosinase zymogen. Based on these sequence data it can be concluded that the polypeptide backbone of the latent form of the tyrosinase PPO4 ranges from Ser2 to Thr565, missing when compared to the gene-derived sequence a small part (46 amino acids) of the C-terminal tail. The high content on hydrophobic amino acids within this missing tail gives rise to speculations whether this part might have a function as a membrane anchor.

Inhibitory and Acceleratory Effects of Inonotus obliquus on Tyrosinase Activity and Melanin Formation in B16 Melanoma Cells.

The aim of the present study is to preliminarily investigate the antimelanogenesis effect of Inonotus obliquus extracts by cell-free mushroom tyrosinase assay. It was found that petroleum ether and n-butanol extracts might contain unknown potential tyrosinase inhibitors, while its ethyl acetate extract might contain some unknown accelerators. Six compounds were isolated and their structures were identified by interpretation of NMR data and nicotinic acid was first discovered in Inonotus obliquus. In cells testing, betulin and trametenolic acid decreased tyrosinase activity and melanin content, while inotodiol and lanosterol significantly increased tyrosinase activity and melanin content, showing an AC⁡50 of 9.74 and 8.43 μM, respectively. Nicotinie acid, 3β,22,25-trihydroxy-lanosta-8-ene, had a little or no effect on tyrosinase. Betulin exhibited a mode of noncompetitive inhibition with a K I = K IS of 0.4 μM on tyrosinase activity showing an IC50 of 5.13 μM and being more effective than kojic acid (6.43 μM), and trametenolic acid exhibited a mode of mixed inhibition with a K I of 0.9 μM, K IS of 0.5 μM, and an IC50 of 7.25 μM. We proposed betulin and trametenolic acid as a new candidate of potent tyrosinase inhibitors and inotodiol and lanosterol as accelerators that could be used as therapeutic agent.