Biochemical characterization of three hamster melanoma variants--I. Tyrosinase activity and melanin content

Tyrosinase activity in the soluble fraction of the cells and melanin content in the whole cells of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) hamster melanomas were studied. The activity of the soluble tyrosinase was highest in MI lower in Ma, and very low in Ab melanoma. Melanin content was greatest in the Ma, lower in MI, and none in Ab melanoma. Acrylamide gel electrophoretic pattern of the soluble tyrosinase consisted of 2 bands in Ma and MI melanomas, and of 1 band in Ab melanoma.     

Association of kinesin and myosin with pigment granules in crustacean chromatophores

Chromatic adaptation in crustaceans results from the differential distribution of colored pigment granules within their chromatophores consequent to cell signaling by neurosecretory peptides. However, the force transducing, mechanochemical protein motors responsible for granule translocation, and their molecular mechanisms of action, are not well understood. The present study uses immunocytochemical techniques and a motility assay in vitro to demonstrate that protein motors from the kinesin and myosin superfamilies are stably associated with membrane-bounded pigment granules in the red, ovarian chromatophores of the freshwater, palaemonid shrimp, Macrobrachium olfersii. Monoclonal antibodies against conventional kinesin heavy chain, and an anti-myosin whole serum, labeled pigment-containing fragments prepared from homogenates of chromatophores with fully dispersed or aggregated pigments: this finding infers a permanent association between the protein motors and the pigment granules, and suggests that such motors may be regulated while bound to their cargos. The pigment aggregator appears to be a myosin since the anti-myosin whole serum attenuated hormonally triggered pigment aggregation in the motility assay in vitro, and induced pigment hyper-dispersion in some chromatophores. Western blots of the chromatophore-containing, ovarian tissue homogenate demonstrated protein bands consistent with myosin II and myosin XII, either of which may be the pigment aggregator. This study provides the first direct evidence for myosin and kinesin protein motors directly and stably associated with pigment granules in crustacean chromatophores, and may represent the first successful isolation of myosin class XII.     

Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus

An optical, ultrastructural, and biochemical study of the melanin accumulation nodules found in the kidney of the teleost fish Sparus auratus is presented. These nodules are randomly distributed in the interstitium of the renal tissue. They are formed by large aggregates of cells replete with melanin granules. The melanin granules occur singly or also in aggregates inside the cells. Most of the granules are electron-dense, but sometimes small electron-lucent spaces within them can be seen. Some secondary lysosomes and dendritic processes can also be observed. Biochemical studies have proved for the first time the existence of measurable tyrosinase activity in those nodules. That activity was assayed using three methods: tyrosine hydroxylation, dopa oxidation, and melanin formation. Furthermore, inhibitors of well-characterized plant and animal skin tyrosinases were effective agents for inhibiting those activities in fish kidney preparations. This finding supports the notion of the existence of true tyrosinase in the melanin accumulation nodules of this tissue. Taking into account the results obtained, the origin and functions of the melanin-containing cells found in the teleost fish kidney are discussed.     

Tyrosinase activity in reversed micelles

The hydroxylase and oxidase activities of mushroom tyrosinase were studied in both sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane and cetyltrimethylammonium bromide (CTAB)/hexane/chloroform reversed micelles. The enzyme presented its highest activity when the water to surfactant molar ratio (W ₀) was 20 for both systems. When entrapped in the AOT reversed micelles, the enzyme activity decreased with the increase in AOT concentration at a constant W ₀, and the enzyme not only presented a higher reaction rate related to its oxidase activity but also a shorter lag period related to its hydroxylase activity. The relation between water activity and W ₀ revealed that enzyme activity in reversed micelles was more related to the size of the micelles which was determined by W ₀ and less to the water activity. Tyrosinase in CTAB reversed micelles showed potential for the analysis of o-diphenols.     

Changes in melanin granules in the fox due to coat color mutations

Hair samples from 11 different coat color phenotypes of the fox (Vulpes vulpes) were examined microscopically to determine the effects of several mutations on melanin granule color and distribution. Standard silver (b/b) causes the production of eumelanin rather than the phaeomelanin produced in wild-type red fox. Fromm brown (bf/bf) and Collicott brown (cb/cb) change the shape of the granules and convert eumelanin to brown and dark brown, respectively. The color dilution of Eastern Pearl (pe/pe) and Mansfield Pearl (pm/pm) is caused by clumping of granules in specific manners. Hairs from animals expressing more than one mutant gene, such as Amber (b/b pe/pe bf/bf), show the color and distribution of granules expected from interactions of independent loci.     

Tyrosinase activity in ionic liquids

Activity of mushroom tyrosinase was studied in three ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF₆]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIm][MeSO₄]), and was compared to that in chloroform. Kinetic parameters of the enzyme were determined and the results indicate that the enzyme in ionic liquids basically follows the same catalytic mechanism as in water, and that the ionic liquids may affect the enzyme activity by direct interacting with the enzyme and thus hindering the E–S binding due to their high hydrophilicity and polarity.     

Tyrosinase in the presumptive pigment cells of ascidian embryos: Tyrosine accessibility may initiate melanin synthesis

Tyrosinase activity appears in the presumptive pigment cells of ascidian embryos (Ciona intestinalis) several hours before the cells begin to synthesize melanin. These presumptive pigment cells develop into the otolith and ocellus pigment cells of the larval brain. Tyrosinase was identified by histochemical tests for tyrosine oxidase and dopa oxidase; both reactions were sensitive to tyrosinase inhibitors. Studies with puromycin suggested that tyrosinase was synthesized at the time it was first detected histochemically and that it was stable during the time interval before melanin synthesis. Supernumerary tyrosinase-containing cells were found adjacent to the presumptive pigment cells in three ascidian species examined (C. intestinalis, Styela partita, and Molgula manhattensis). Tyrosinase disappeared from the supernumerary pigment cells during larval development and these cells did not synthesize melanin.Tyrosinase in the presumptive and supernumerary pigment cells is apparently a functional enzyme which does not interact with substrate. External substrates ( -tyrosine and -dopa) did not react with enzyme in the living cells before the normal time of pigment synthesis, but gentle disruption of the cells (by freezing-and-thawing or osmotic shock) released active tyrosinase. Progessive enlargement of nonpigmented vesicles in the otolith cells of embryos exposed to phenylthiourea, an inhibitor of tyrosinase activity, suggested that tyrosinase vesicles actively accumulate tyrosine at the beginning of melanin synthesis. This tyrosine accumulation probably initiates melanin synthesis.     

The regulation of motile activity in fish chromatophores

Chromatophores, including melanophores, xanthophores, erythrophores, leucophores and iridophores, are responsible for the revelation of integumentary coloration in fish. Recently, blue chromatophores, also called cyanophores, were added to the list of chromatophores. Many of them are also known to possess cellular motility, by which fish are able to change their integumentary hues and patterns, thus enabling them to execute remarkable or subtle chromatic adaptation to environmental hues and patterns, and to cope with various ethological encounters. Such physiological color changes are indeed crucial for them to survive, either by protecting themselves from predators or by increasing their chances of feeding. Sometimes, they are also useful in courtship and mutual communications among individuals of the same species, leading to an increased rate of species survival. Such strategies are realized by complex mechanisms existing in the endocrine and/or nervous systems. Current studies further indicate that some paracrine factors such as endothelins (ETs) are involved in these processes. In this review, the elaborate mechanisms regulating chromatophores in these lovely aquatic animals are described.     

Tyrosinase activity and abundance in Cloudman melanoma cells

Rabbit anti-tyrosinase antibodies were used to study the abundance, processing, and degradation of tyrosinase in murine (Cloudman) melanoma cells. The polyclonal antibodies precipitated low-molecular-weight (68,000 and 70,000) and high-molecular-weight (78,000 and 80,000) tyrosinases that had a precursor-product relationship. Cells with high basal tyrosinase activity had high levels of newly synthesized tyrosinase. Cells with low tyrosinase activity synthesized less tyrosinase and degraded the enzyme at a faster rate than cells with high tyrosinase activity. Melanotropin (melanocyte stimulating hormone), dibutyryl cyclic adenosine monophosphate, and isobutylmethylxanthine caused an increase in the abundance of newly synthesized tyrosinase that was directly proportional to the increase in enzyme activity. This enzyme was not a phosphoprotein. Other changes in the culture conditions that increased the level of tyrosinase activity increased the abundance of newly synthesized enzyme. It is thus concluded that the level of tyrosinase activity in Cloudman melanoma cells is a direct reflection of the abundance of enzyme protein.     

Tyrosinase activity and the expression of the agouti gene in the mouse

Tyrosinase activity at the time of phaeomelanin synthesis in neonatal mice is lower in agouti than in black skin and hair bulb tissue, and this depressed activity is associated with a reduction in the electrophoretically distinct de novo form of the enzyme. Direct chemical measurements of sulphydryl compounds show elevated levels in agouti hair bulb tissue at this stage of development. The addition of exogenous copper to hair bulb extracts raises the activity of tyrosinase in agouti to approximately the black level but has no affect on black itself. These results are discussed in relation to the role of sulphydryl compounds and copper availability in regulating tyrosinase activity and turnover.     

Tyrosinase activity in the first coat of agouti and black mice

Tyrosinase activity was compared in the skin and hair bulbs of young black and agouti mice between 4 and 12 days old. Differences in activity were found to be maximal in both the hair and skin at the time of yellow pigment synthesis in agouti mice. Histological examination suggested that the number of dopa-positive melanocytes is similar in the hair bulbs of agouti and black mice. The level of SH-compounds in the hair bulb was examined and found to be elevated in agouti tissue at the time of phaeomelanin formation. It was shown that sulphydryl compounds such as cysteine and glutathione have an inhibitory effect on tyrosinase, and it is possible that the elevated levels of SH-compounds are responsible for a reduction in tyrosinase activity in agouti mice. In agouti hair bulbs, this effect can be reversed in vitro by addition of copper.     

Pigment pattern formation in the quail mutant of the silkworm, Bombyx mori: parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes

The larval pigment pattern in the silkworm, Bombyx mori, is formed by melanin, ommochromes and pteridines. During development all these pigments are synthesized autonomously, and possibly also with mutual interaction between them, to yield unique pigment patterns. In order to find the key trigger for such pigment pattern formation, developmental changes in pteridine biosynthesis were studied using the quail mutant (q/q), which has darker larval marks formed by melanin and an abundance of ommochromes in the integument. In the current study, emphasis has been placed on the analysis of GTP-cyclohydrolase I (GTP-CH I), which is a key enzyme for the biosynthesis of pteridines, during the development of the silkworm. Results of Northern blotting showed that in the quail mutant strong signals of GTP-CH I mRNA appeared around each period of ecdysis, while no such signals appeared in the background strain (+q/q) used. Also, both GTP-CH I activities and pteridine content were higher in the quail mutant compared with the background strain. These results strongly suggest that pteridine biosynthesis is closely linked to the formation of melanin and ommochromes. It is also suggested here that in the silkworm a recessive gene (q) may be involved in the regulation of its pigment pattern formation.