Role In Melanogenesis Research Articles

A mass spectrometric investigation on the possible role of tryptophan and 7‐hydroxytryptophan in melanogenesis

The activity of tyrosinase and peroxidase + H2O2 in promoting melanogenesis from tryptophan (Trp) and 7-hydroxytryptophan (7-HTP) has been investigated. The reaction samples have been drawn at different reaction times and analysed by MALDI mass spectrometry. The data obtained showed that tryptophan undergoes, under tyrosinase and peroxidase action, an oligomerization process mainly due to the reaction of anthranilic acid (AA) and Trp. However, analysing the UV and fluorescence data, it is seen that the oligomers cannot belong to the melanin pattern, but their possible role in melanogenesis is not to be excluded. Once it reacts with the two enzymes, 7-hydroxytryptophan leads to dark brown products, indicating its possible role in melanin production. In contrast to what was observed in the case of 5-hydroxytryptophan, for which oligomers were constituted by 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT) units, the MALDI data indicate a sharply different behaviour for 7-HTP. In fact, in the case of 5-hydroxytryptophan, oligomerization takes place through the formation of 5-hydroxytryptamine and the oligomerization products are due to mixed 5-HTP-5-HT oligomers. In the case of 7-hydroxytryptophan, the formation of 7-hydroxytryptamine (7-HT) is also observed, but it does not seem to play any role; the only oligomerization products formed are due to the reaction of 7-hydroxytryptophan and AA. The data so obtained indicate that 7-hydroxytryptophan acts like an effective melanin precursor in the presence of both tyrosinase and peroxidase + H2O2.

MART-1 Is Required for the Function of the Melanosomal Matrix Protein PMEL17/GP100 and the Maturation of Melanosomes

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.

Role and modulation of tyrosinase/tyrosinase related protein‐1 complex and PKC beta‐I in melanogenesis

Role and modulation of tyrosinase/tyrosinase related protein‐1 complex and PKC beta‐I in melanogenesis

Immunohistochemical determination of HER-2/neu, c-Kit (CD117), and vascular endothelial growth factor (VEGF) overexpression in malignant melanoma.

To determine the prevalence and evaluate the possible prognostic value of the molecular targets in malignant melanoma, we studied the overexpression of HER-2/neu, c-Kit, and vascular endothelial growth factor (VEGF) in this patient population. Overexpression of HER-2/neu, c-Kit, and VEGF was evaluated using immunohistochemical assays in 202 archival tissue specimens. Only two patients (0.9%) revealed HER-2/neu overexpression, whereas 46 (22.8%) revealed c-Kit and 42 (20.8%) specimens showed VEGF overexpression. Multivariate analysis performed did not show a significant difference in survival between c-Kit-positive and c-Kit-negative groups (P = 0.36) and VEGF-positive and VEGF-negative groups (P = 0.25). Interestingly, c-Kit was more likely to be overexpressed in the superficial spreading type and VEGF was overexpressed preferentially in the amelanotic melanoma type. HER-2/neu has no role in melanogenesis. Both c-Kit (expressed in superficial spreading disease) and VEGF (expressed in amelanotic melanoma) may have significant therapeutic implications as molecular targets, which warrants further investigation.

Characterization of the human RAB38 and RAB7 genes: exclusion of new major pathological loci for Japanese OCA

Background: Oculocutaneous albinisms (OCAs) are due to various gene mutations that cause a disruption of melanogenesis in the melanocyte. Four different genes associated with human OCA have been reported, however, not all of OCA patients can be classified according to these four genes. We have sought to find a new major locus for Japanese OCA. Recently two genes, RAB38 and RAB7, were reported to play an important role in melanogenesis in the melanocyte, suggesting that these two genes could be good candidates for new OCA loci. Objective: To determine the structures of the human RAB38 and RAB7 genes, and examine if the two genes are new major loci for Japanese OCA. Methods: We screened mutations in these genes of 25 Japanese OCA patients who lacked mutations in the OCA1 and OCA2 genes with SSCP/heteroduplexes method. Results: We determined the both genes, and their genomic organizations to design the primers for SSCP/heteroduplexes method. And then we screened mutations, but no mutation was detected. Conclusion: Neither of the genes is a new major locus for Japanese OCA.

Sphingosine-1-phosphate decreases melanin synthesis via sustained ERK activation and subsequent MITF degradation.

Sphingosine-1-phosphate (S1P) has emerged as a bioactive lipid modulator that mediates a variety of cell functions. However, the effects of S1P on melanogenesis are not well known. Therefore, we investigated the actions of S1P on melanin synthesis using a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. This study shows that S1P significantly inhibits melanin synthesis in a concentration-dependent manner, and also that the activity of tyrosinase was reduced in S1P-treated cells. In contrast, a specific extracellular signal-regulated protein kinase (ERK) pathway inhibitor, PD98059, increased tyrosinase activity and melanin production, and PD98059 also restored the S1P-induced reduction of tyrosinase activity and pigmentation. In addition, we found that S1P induces the sustained activation of ERK and the subsequent degradation of microphthalmia-associated transcription factor (MITF), which plays a key role in melanogenesis. Thus, we further studied the relationship between the ERK pathway and melanin synthesis. PD98059 was found to prevent the S1P-induced MITF phosphorylation and degradation and to abrogate the S1P-induced downregulation of tyrosinase and of tyrosinase-related protein 1 (TRP1) production. These results indicate that the ERK pathway is potently involved in the melanogenic signaling cascade, and that S1P-induced ERK activation contributes to reduced melanin synthesis via MITF degradation. Therefore, we suggest that S1P reduces melanin synthesis by ERK activation, MITF phosphorylation and degradation, and by the subsequent downregulation of tyrosinase and TRP-1 production.

Immunohistochemical determination of HER-2/neu overexpression in malignant melanoma reveals no prognostic value, while c-Kit (CD117) overexpression exhibits potential therapeutic implications

BackgroundHER-2/neu and c-kit (CD117) onco-protein are increasingly being recognized as targets for therapy in solid tumors, but data on their role in malignant melanoma is currently limited. We studied the prevalence of overexpression of HER-2/neu and c-Kit in 202 patients with malignant melanoma to evaluate a possible prognostic value of these molecular targets in malignant melanoma.MethodsOverexpression of HER-2/neu and c-Kit was evaluated using immunohistochemical assays in 202 archival tissue specimens.ResultsBetween 1991 and 2001, 202 subjects (109 males; 54% and 93 females; 46%) with malignant melanoma were studied with a mean age of 57 years (age range: 15–101 years). The most common histologic type was amelanotic melanoma (n = 62; 30.7%) followed by superficial spreading melanoma (n = 54; 26.7%). The depth of penetration of melanoma (Breslow thickness, pT Stage) ranged from 0.4 mm (stage pT1) to 8.0 mm (stage pT4A). Mean thickness was 2.6 mm (stage pT3A). The ECOG performance scores ranged from 0 to 3. Only 2 patients (0.9%) revealed HER-2/neu overexpression, whereas 46 (22.8%) revealed c-Kit overexpression. Multivariate analysis performed did not show a significant difference in survival between c-Kit positive and negative groups (p = 0.36). Interestingly, not only was c-Kit more likely to be overexpressed in the superficial spreading type, a preliminary association between the presence or absence of c-Kit overexpression and the existence of another second primary tumor was also observed.ConclusionsThe results of our large study indicate that the HER-2/neu onco-protein neither has a role in melanogenesis nor is a potential target for clinical trials with monoclonal antibody therapy. This indicates there is no role for its testing in patients with malignant melanoma. Although c-Kit, expressed preferentially in the superficial spreading type, may not have prognostic value, it does have significant therapeutic implications as a molecular target warranting further investigation.

Gamma-glutamyl transpeptidase and its role in melanogenesis: redox reactions and regulation of tyrosinase.

Metabolism of glutathione by gamma-glutamyl transpeptidase (gamma-GT) at the level of cell membrane has been shown to generate hydrogen peroxide in many cell types including human melanomas. gamma-GT does not appear to be involved in cysteine uptake for pheomelanin production in melanoma cells and does not contribute significantly to the pheomelanin synthesized in B16 melanoma cells. We have therefore examined the possibility of gamma-GT mediated production of prooxidant reactions and its effect, if any, on pigmentation using B16 melanoma cells. Our results indicate that in B16 melanoma cells, gamma-GT activity leads to the production of hydrogen peroxide. We further show that the nuclear levels of the redox sensitive transcription factor NF-kappa B is regulated by H2O2 formed by the action of gamma-GT: stimulation and inhibition of gamma-GT affect the levels of NF-kappa B. Tumor necrosis factor alpha, a hypopigmenting cytokine, known to activate NF-kappa B also up-regulates the gamma-GT messenger RNA and activity. Stimulation of gamma-GT generated prooxidant reactions led to a decrease in tyrosinase activity. We therefore propose that prooxidant reactions mediated by gamma-GT in turn regulate the levels of tyrosinase in pigment cells. Our findings thus introduce a new aspect in the regulation of pigmentation and ascribe a novel role for gamma-GT in pigment cells.

Induction and characterization of mutations at the b locus of the medaka, Oryzias latipes.

The b locus is one of the most familiar pigmentation loci in the medaka, but its biochemical function is still unknown. Here we report induction of new mutations at the b locus by radiation and ENU. We also characterized all these mutations and previously isolated spontaneous ones on the phenotypic basis. Unexpectively, all the 18 induced mutations reduced melanin contents in both eyes and skin correlatively, although degree of reduction was varied from mutations to mutations. Moreover, presumed null mutants (bs8, bg8, bc2, bd3, bd6, bg13, bg19, bg24) had slightly melanized (dark red) eyes. These results suggest that the b-locus product plays an important but not a critical role in melanogenesis. The spontaneous mutants were divided into two types: one (bdl2, bdl3, and bp) had similarities with the induced mutants in that they had slightly colored eyes and skin, the other (bv, B', bd, bdl1, and b) exhibited normally black eyes but lightly colored skin. The present study supports our recent results (Fukamachi et al., 2001) that mutational changes were found in the coding region of the b gene in some of the mutants which reduced both eyes and skin melanogenesis, while the mutational change for the b allele could not be found there. We speculate that the bv, B', bd, bdl1, and b alleles might arise by the mutations in the regulatory region for skin melanogenesis.

Over-expression of MSG1 transcriptional co-activator increases melanin in B16 melanoma cells: a possible role for MSG1 in melanogenesis.

MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase.

Syntaxin 7 Complexes with Mouse Vps10p Tail Interactor 1b, Syntaxin 6, Vesicle-associated Membrane Protein (VAMP)8, and VAMP7 in B16 Melanoma Cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Characterization of mutants defective in melanogenesis and a gene for tyrosinase of Streptomyces griseus.

Nitrosoguanidine-induced melanin-negative mutants of Streptomyces griseus were classified according to their ability to produce streptomycin and A-factor, and to form aerial mycelium. A large proportion of the mutants showed deficiency in either antibiotic production and morphological development or both, suggesting close regulatory correlation between melanogenesis and morphological and physiological differentiation. The tyrosinase-encoding mel operon of S. griseus was cloned and examined for its role in melanogenesis of this organism. As in other Streptomyces homologues, the operon consisted of two open reading frames, melC1 encoding the putative cofactor and melC2 encoding the tyrosinase. Regardless of the distinct sequence similarity, introduction of the operon on plasmids failed to confer melanin production in the melanin-negative mutants, and the disruption of melC2 barely affected the melanin productivity, which indicated the presence of another enzyme involved in the melanogenesis in S. griseus. On the other hand, mel on a high-copy-number plasmid caused precocious aerial mycelium formation in Streptomyces lividans TK21 suggesting a stimulatory role of tyrosinase in morphological development.

New insights on the structure of the mouse silver locus and on the function of the silver protein.

The melanosomal proteins encoded by the silver locus play important roles in melanogenesis. The human locus yields two proteins, PMEL17 and GP100, by alternative mRNA splicing. The mouse si locus was reported to encode a Pmel17 protein, and later gp87, a GP100 homologue. When we re-examined the products of wild-type and silver-mutant mouse si loci, RT-PCR of wild-type RNA and genomic DNA sequence accounted for gp87 but excluded the occurrence of Pmel17. Analysis of cDNA from the silver (si/si) melanocyte line, melan-si, showed that the pathogenic mutation is a G to A substitution at nt 1808, which yields a premature stop codon and a predicted protein truncated in the C-terminus. This was confirmed by reaction of a specific anti-gp87 antiserum with si/si melanocyte extracts. To further explore gp87 function, we compared the DHICA oxidase activity of extracts from B16, melan-si (heterozygous for the brown mutation and homozygous for the silver mutation) and Cloudman S91 cells (homozygous for the brown mutation), since both TRP1 and gp87 are thought to be involved in DHICA oxidation/polymerization. Cloudman extracts do not oxidize significantly DHICA and its methyl ester, supporting the involvement of native mouse TRP1 in DHICA oxidation. Extracts from B16 and melan-si do not show significant differences for the oxidation of free acid and methylated dihydroxyindoles, indicating that the mechanism is not decarboxylative. Melan-si extracts are very efficient in catalyzing dihydroxyindole oxidation, in spite of being heterozygous for the TRP1 mutation, consistent with a stablin effect for the wild-type gp87 protein. On the other hand, aggregated and degraded forms of that mutant gp87 protein are found in the cytosolic fraction of melan-si, suggesting that misrouting and aberrant processing of the gp87 and tyrosinase may also be related to the high DHICA oxidase activity of these melanocytes.

Regulation of the murine silver locus product (gp87) by the hypopigmenting cytokines TGF-beta1 and TNF-alpha.

The melanosomal proteins encoded by the silver (si, SILV, or PMEL17) locus play important roles in melanogenesis and are actively investigated as targets for melanoma immunotherapy. The human silver locus yields two proteins, gp100 and PMEL17, by alternative splicing of a common mRNA precursor. Mouse melanocytes exclusively express the gp100 orthologue, here termed gp87, thus providing a simpler model with which to study the silver locus products. We have analyzed the effects of [Nle4, D-Phe7]-alpha melanocyte-stimulating hormone (alphaMSH) and two hypopigmenting cytokines, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, on the expression of gp87 in B16 mouse melanoma cells. TNF-alpha and TGF-beta1 (at saturating doses for 48 hr) decreased gp87 mRNA by 50%. The gp87 protein was almost undetectable by Western immunoblotting after TNF-alpha treatment, but was not affected by TGF-beta1. alphaMSH increased the mRNA and the gp87 protein approximately 2-fold. Moreover, the amount of gp87 was not reduced by TNF-alpha in the presence of the hormone, in spite of a 50%, decrease in its mRNA. Therefore, the levels of mRNA and gp87 protein did not correlate after treatment with the cytokines. Overall, our data suggest that the silver locus product is not regulated exclusively at the transcriptional level, and highlight the importance of still-uncharacterized regulatory translational and/or post-translational events.

Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses.

The Kit receptor tyrosine kinase functions in hemato- poiesis, melanogenesis and gametogenesis. Kit receptor-mediated cellular responses include proliferation, survival, adhesion, secretion and differentiation. In mast cells, Kit-mediated recruitment and activation of phosphatidylinositol 3'-kinase (PI 3-kinase) produces phosphatidylinositol 3'-phosphates, plays a critical role in mediating cell adhesion and secretion and has contributory roles in mediating cell survival and proliferation. To investigate the consequences in vivo of blocking Kit-mediated PI 3-kinase activation we have mutated the binding site for the p85 subunit of PI 3-kinase in the Kit gene, using a knock-in strategy. Mutant mice have no pigment deficiency or impairment of steady-state hematopoiesis. However, gametogenesis is affected in several ways and tissue mast cell numbers are affected differentially. While primordial germ cells during embryonic development are not affected, Kit(Y719F)/Kit(Y719F) males are sterile due to a block at the premeiotic stages in spermatogenesis. Furthermore, adult males develop Leydig cell hyperplasia. The Leydig cell hyperplasia implies a role for Kit in Leydig cell differentiation and/or steroidogenesis. In mutant females follicle development is impaired at the cuboidal stages resulting in reduced fertility. Also, adult mutant females develop ovarian cysts and ovarian tubular hyperplasia. Therefore, a block in Kit receptor-mediated PI 3-kinase signaling may be compensated for in hematopoiesis, melanogenesis and primordial germ cell development, but is critical in spermatogenesis and oogenesis.

Identification of a Novel Isoform of Microphthalmia-Associated Transcription Factor That Is Enriched in Retinal Pigment Epithelium

Mutations at the mouse locus encoding microphthalmia-associated transcription factor (Mitf) affect the development of many cell types, including retinal pigment epithelium (RPE), melanocytes, mast cells, and osteoclasts. Here we have identified a novel Mitf isoform, Mitf-a, and its human homologue MITF-A by cDNA cloning. MITF-A consists of 520 amino acid residues and differs in the amino-terminus from authentic melanocyte-type MITF (MITF-M). MITF-A mRNA is widely expressed and represents a predominant MITF isoform in cultured RPE cells, whereas MITF-M mRNA is exclusively expressed in melanocytes and melanoma cells. In situ hybridization analysis suggested that Mitf-a mRNA is enriched in the prospective RPE of mouse embryo. Moreover, transient cotransfection assays suggested that MITF-A activated transcription of the tyrosinase and tyrosinase-related protein 1 genes. MITF-A/Mitf-a therefore may play an important role in melanogenesis in RPE.

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: a projected basis for immunotherapy.

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Delta1b and Delta1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Delta1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Delta1d reached 4.0% as compared with 0. 8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Delta1b and Delta1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Autocrine Regulation of Neural Crest Cell Development by Steel Factor

Steel factor (SLF) and its cognate receptor, c-kit, have been implicated in the generation of melanocytes from migrating neural crest (NC) cells during early vertebrate embryogenesis. However, the source of SLF in the early avian embryo and its precise role in melanogenesis are unclear. We report here that NC cells themselves express and release SLF protein, which in turn acts as an autocrine factor to induce melanogenesis in nearby NC cells. These results indicate that NC cell subpopulations play an active role in the determination of their cell fate and suggest a different developmental role for the embryonic microenvironment than what has been previously proposed.

Expression of c-kit and kit ligand proteins in normal human tissues.

The c-kit receptor and its cognate ligand, KL, play a critical role in melanogenesis, gametogenesis, and hematopoiesis. Studies on the expression of c-kit and KL have been primarily focused on mouse development. We undertook the present study to characterize the pattern of expression of these molecules in normal adult human tissues. Using immunohistochemistry and consecutive tissue sections from the same block, we evaluated a variety of well-preserved normal tissues for c-kit and KL microanatomic distribution. c-kit protein was identified in tissue mast cells, melanocytes, glandular epithelial cells of breast, parotid, dermal sweat, and esophageal glands. Scattered c-kit immunoreactivity was also observed for testicular and ovarian interstitial cells. A striking regional distribution of c-kit was detected in the central nervous system, particularly in the cerebellum, hippocampus, and dorsal horn of the spinal cord. KL protein was identified in cells complementary to staining for the receptor, such as glandular myoepithelium of breast and sweat glands. Intense KL immunoreactivity was observed in smooth muscle cells of the bladder, cervix, uterus, and gastrointestinal tract, as well as in striated and cardiac muscle. Strong KL staining was also detected in prostate fibromuscular stroma cells. In the central nervous system, KL expression was confined to Golgi and Purkinje cells in the cerebellum. These results suggest a role for this receptor and its ligand in the maintenance of a variety of fully differentiated tissues.

A new enzymatic function in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1 (TRP1).

Since the characterization of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as a major melanogenic intermediate, the fate of this compound and the mechanisms of its incorporation into the melanin polymer have become major issues in the study of melanogenesis. DHICA is a stable dihydroxyindole with a low rate of spontaneous oxidation, suggesting that enzymatic mechanism(s) might contribute to its evolution. The most obvious candidates are the melanosomal tyrosinases. We have recently shown that mouse melanosomes contain two electrophoretically distinct tyrosinase isoenzymes, termed low electrophoretic mobility tyrosinase (LEMT) and high electrophoretic mobility tyrosinase (HEMT), that can be resolved and purified. In this study, we report immunological evidence indicating that LEMT corresponds to the protein encoded by the brown locus (termed tyrosinase-related protein-1, TRP1), while HEMT corresponds to the tyrosinase encoded by the albino locus. We have compared the ability of both isoenzymes to catalyze DHICA evolution as determined by high performance liquid chromatography; although LEMT is a relatively poor tyrosine hydroxylase and DOPA oxidase as compared to HEMT, it was readily able to accelerate DHICA consumption concomitant with the production of a brownish product. However, the DHICA conversion activity of HEMT was barely detectable. The ability of purified LEMT to catalyze DHICA conversion could be almost completely abolished by treatment with heat or trypsin, and was inhibited in a concentration dependent way by the tyrosinase inhibitor 2-phenylthiourea and by L-tyrosine. Moreover, in the presence of low concentration of ascorbate, the DHICA conversion activity of LEMT displayed a lag period which was progressively longer at higher ascorbate concentrations. Based on the relationship between ascorbate added, enzyme activity, and lag period, it is very likely that the DHICA converting activity is indeed a DHICA oxidase activity. This was further proven by the demonstration that the product reacts rapidly and efficiently with the quinone trapping reagent 3-methyl-2-benzothiazolinone hydrazone, yielding a colored adduct similar to the one obtained with DOPAquinone. The DHICA oxidase activity of LEMT displayed a Km for DHICA of about 0.8 mM, as compared to 1.9 mM for L-DOPA and 0.23 nM for L-tyrosine. These results suggest that TRP1, the product of the brown locus, is indeed a tyrosinase with DHICA oxidase activity. However, as opposed to the tyrosinase encoded by the albino locus, TRP1's role in melanogenesis could be more directly related to DHICA metabolism than to the first steps of the pathway.