Tyrosinase Protein Is Expressed Also in Some Neural Crest Derived Cells Which Are Not Melanocytes

Some neural crest cells give rise to pigment cells in early ontogenesis. We tested here whether tyrosinase-a key enzyme in melanogenesis—was present in some nonpigment neural crest derivatives in adult hamsters. Interestingly enough, inactive tyrosinase protein was detected, using indirect immunofluorescence, in the satellite cells of spinal ganglia and Schwann cells of sciatic and facial nerves in normal adult animals. The results of cell blotting from spinal ganglia were similar to the fluorescence findings. Thus, our results seem to support the hypothesis that Schwann cells, satellite cells of spinal ganglia, and melanocytes may be more intimately related developmentally than other neural-crest-derived cells. Moreover, since we detected tyrosinase protein in cells which normally do not produce melanosomes, it could be deduced that, during the melanocyte's differentiation from its cell precursor, the expression of tyrosinase protein might precede the point when melanosomes begin to differentiate from known cytoplasmic structures.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Tyrosinase Isoenzymes: Two Melanosomal Tyrosinases With Different Kinetic Properties and Susceptibility to Inhibition by Calcium

Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS-PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and dopa oxidase activity and were stereospecific and sensitive to phenylthiourea, proving that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme, Immunoprecipitation and Western blots performed with the specific antibody αPEP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were noncompetitively inhibited by Ca²⁺ at physiologically relevant concentrations. However, the b protein was apparently more susceptible, since maximal inhibition was reached at lower Ca²⁺ concentrations for LEMT. Moreover, binding of Ca²⁺ to the tyrosinases resulted in a noticeable thermal destabilization of the enzymes, which was also more pronounced for LEMT.     

The Presence of Tyrosinase and Related Proteins in Human Epidermis and Their Relationship to Melanin Type

The present study was carried out to investigate the abundance of tyrosinase and related proteins (TRP-1 and TRP-2) in human epidermis and their relationship to melanin type. Positive immunocytochemical staining was seen for all three proteins in epidermal melanocytes. For each protein the numbers of positively stained melanocytes were similar in all subjects studied irrespective of skin type. Following 5 daily suberythemal doses of UVB the melanocytes were larger, more dendritic, and increased in number. With TRP-1 and TRP-2 the increase in number in response to UVB was unrelated to skin type and, hence, with melanin type but with tyrosinase there was a much greater increase in skin types III and IV than in skin type I and II. The enhanced numbers of tyrosinase-positive melanocytes were accompanied by increased staining intensity, suggesting a greater expression of tyrosinase in the melanocytes from skin types III and IV compared with skin types I and II. This increase in tyrosinase could be related to the greater levels of eumelanin found in skin types III and IV, and this is in keeping with the view that higher levels of tyrosinase are associated with the production of eumelanin than phaeomelanin.     

Fine Structure of Melanogenesis in the Ink Sac of Sepia offidnalis

The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane-bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 μm and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.     

Human TRP-1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of ³H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.     

Tumor Suppressor p53 Down-Regulates Tissue-Specific Expression of Tyrosinase Gene in Human Melanoma Cell Lines

Tyrosinase, the key gene in melanin pigment synthesis, is tissue-specifically expressed in melanocytic cells. Expression of this gene is regulated by various hormones, carcinogens, and environmental factors. The molecular basis underlying tyrosinase gene regulation is still not clear. In this report, we present the effects of tumor suppressor p53 protein on tyrosinase gene expression and melanin synthesis in human melanoma. After stable transfection of wild type p53 expression plasmid into a highly pigmented melanoma cell line, overexpression of wt p53 suppressed the pigmentation of the melanoma cells. The loss of pigmentation was associated with the loss of endogenous tyrosinase expression at the activity and mRNA levels. In order to determine whether the p53 repression of tyrosinase mRNA involved modulation of tyrosinase promoter activity, transient transfection approaches involving p53 expression plasmid and construct containing chloramphenicol acetyl transferase (CAT) reporter gene linked to 270 bp tissue-specific tyrosinase promoter have been used. p53 specifically repressed CAT gene expression from the tyrosinase promoter and not from the Rous sarcoma virus promoter. These data suggest that in human melanoma p53 down-regulates the tissue-specific expression of tyrosinase gene and subsequent melanin synthesis.     

Mutations affecting pigmentation and shape of the adult zebrafish

 Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish. Received: 17 June 1996 / Accepted: 15 July 1996     

l-DOPA Is a Substrate for Tyrosine Hydroxylase

Abstract: In the presence of thiols, tyrosine hydroxylase (TH) oxidizes l -dihydroxyphenylalanine ( l -DOPA) with a specific activity of up to 140 nmol min⁻¹ mg⁻¹ at 37°C and pH 7.0, which is ∼12–50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2-mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of l -DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.