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At least 19 recordsLinked to original sources
PubMed1977

Granule-bound tyrosinase: solubilization and its relation to the soluble form of tyrosinase.

Disc gel electrophoresis of tyrosinase solubilized by sodium deoxycholate (DOC) from the particle fraction of B16 mouse melanoma was carried out in the presence of DOC. A single tyrosinase band, considered to be T3, was detected at the Rx value of 0.60 against Coomassie brilliant blue. The T3 band which is located between T1 and T2 seems to be distinct from the soluble forms of tyrosinase. Its molecular weight was estimated to be 102,000. When treated with proteolytic enzymes or refrigerated, the T3 molecule converted into a molecule whose mobility was equivalent tothat of T1.

Animals↗
PubMed1977

Detection of heterozygotes for tyrosinase-negative oculocutaneous albinism by hairbulb tyrosinase assay.

Five heterozygotes for ty-neg OCA had absent or markedly reduced hairbulb tyrosinase activity, when compared to normally pigmented control subjects. Clinically, all five were fully pigmented and could not be distinguished from the normal control subjects. It is proposed that the quantitative hairbulb tyrosinase assay can be used to detect heterozygotes for ty-neg OCA. A mechanism to explain the low levels of activity in the hairbulbs from heterozygotes is discussed.

Albinism↗
PubMed1975

Immunochemical studies on tyrosinase induction in Neurospora.

An immunoassay for tyrosinase, using the modified bacteriophage technique, was developed: Tyrosinase of Neurospora was conjugated to bacteriophage T4 using glutaraldehyde as a cross-linking agent. The conjugated phage that survived the coupling process could be inactivated by antiserum raised in rabbits against pure tyrosinase, but not by normal serum. This inactivation was specifically inhibited by pure Neurospora tyrosinase, and the degree of inhibition was proportional to the concentration of tyrosinase within the range of 30-150 ng/ml. Crude mycelial extract possessing tyrosinase activity could similarly inhibit the inactivation of the conjugated phage by the antiserum. To evaluate the tyrosinase content of crude extracts their inhibitory capacity was compared to that of known amounts of pure tyrosinase, and the amounts thus calculated agreed with those predicted from an enzymatic assay. The tyrosinase-bacteriophage immunoassay was used for the quantitation of tyrosinase-antigen in crude extracts of Neurospora cultures that had been induced to form tyrosinase by the addition of ethionine. Enzymatic activity appeared after a lag of several hours, increased for 2-3days and then declined. Immunological assays of these cultures showed: (a) serologically reactive protein started to accumulate upon culture starvation and was evident during the lag period; (b) specific activity (units per mg antigen) was constant throughout induction; (c) at the phase of decrease in mycelial enzyme content, increasing amounts of serologically reactive protein were detected in the medium, indicating that some enzyme was eventually excreted. These results show that the lag is not a qualitatively distinct period, and support the previously forwarded notion that tyrosinase is synthesized de novo upon induction.

Antigen-Antibody Reactions↗
PubMed1992

Mutations of the tyrosinase gene in oculocutaneous albinism.

Since our first report showing that the phenotype of tyrosinase-negative or type IA oculocutaneous albinism (OCA) is a consequence of a mutation in the tyrosinase gene (Tomita et al., Biochem. Biophys. Res. Commun., 164:990-996, 1989), a number of mutations were found in the tyrosinase gene of OCA patients. However, to establish the molecular basis of OCA in each patient, we must carry out several important experiments as summarized here. First, we should confirm that the cloned or amplified genomic DNA segments are not derived from the pseudogene or related gene. It should be noted that the putative tyrosinase pseudogene contains the sequence almost identical to exons 4 and 5, including their exon/intron boundaries of the authentic tyrosinase gene. Thus, the mutations, detected in exon 4 or 5 amplified from genomic DNA, must be carefully analyzed to exclude a possibility that the mutation is located in the pseudogene. Second, it is of significance to confirm the promoter activity of the patients' tyrosinase gene. Accordingly, we established the cell-free transcription system derived from melanoma cells where the cloned tyrosinase gene is faithfully transcribed. Finally, transient expression assay of mutant tyrosinase is invaluable to conclude that OCA phenotypes are associated with the mutant tyrosinase alleles. I also discuss the implications of a cluster of mutation sites in exon 1 coding for the amino-terminus of tyrosinase.

Albinism, Oculocutaneous↗
PubMed1992

Tyrosinase and the regulation of coat color changes in C3H-HeAvy mice.

Hair follicular tyrosinase activity was measured during hair growth in neonatal, pubertal, and adult C3H-HeAvy mice that show differences in coat color as a result of changes in the synthesis of eumelanin and pheomelanin. Tyrosinase activity increased during hair growth in all mice but higher levels were found at puberty, when the mice grow a dark, eumelanin coat of hair, than during early and adult life, when the hair follicular melanocytes produce mainly pheomelanin. This suggests that tyrosinase is more important for the synthesis of eumelanin than that of pheomelanin. The increased tyrosinase activity associated with eumelanogenesis in the pubertal mice could not be explained by enhanced transcription of the tyrosinase gene or enzyme synthesis and appeared to be the result of a post-translational activation. Such an activation of tyrosinase was lacking in the neonatal and adult mice; in the latter this was accompanied by a reduction in the glycosylation of tyrosinase and the proportion of enzyme associated with the melanosomal fraction. Our findings suggest that post-translational mechanisms are important control points in the regulation of tyrosinase and that differences in their level of activation are responsible for determining the patterns of melanogenesis in the C3H-HeAvy mice, but it is still not clear how these mechanisms are regulated. Although cyclic AMP increased tyrosinase synthesis it had no post-translation activating effect. The neonatal mice, unlike their pubertal and adult counterparts, also lacked dopachrome converting activity and TRP tyrosinase-related protein-1 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Formation of melanin-tyrosinase complex and its possible significance as a model for control of melanin synthesis.

Previous reports have shown that the tyrosinase activities of melanosomes and soluble tyrosinase isolated from melanoma were diminished when these preparations were incubated with 3,4-dihydroxyphenylalanine (dopa). It was concluded from these results that the tyrosinase activities of these preparations decreased when melanin was synthesized in these systems. The present investigation has revealed that melanin synthesized in vitro from dopa formed a complex with purified mushroom tyrosinase. The addition of melanin diminished the tyrosinase activity of the sample. These results show that the formation of the melanin-tyrosinase complex results in a decrease in the activity of the tyrosinase solution. The tyrosinase activity of the melanin-tyrosinase complex could not be increased by certain procedures which were previously found to enhance the tyrosinase activity of isolated melanosomes.

Catechol Oxidase↗
PubMed1992

Conserved regulatory mechanisms of tyrosinase genes in mice and humans.

In vertebrates, melanin production is restricted to pigment cells. This cell type-specific melanogenesis is considered to involve cell type-specific expression of the tyrosinase gene. Recently, there have been several reports that sequences in the 5' flanking region of the mouse tyrosinase gene are responsible for cell type-specific expression of the transgene in mice. As the first step in the study of the evolution of the regulatory mechanisms for tyrosinase gene function in vertebrates, we constructed a fused gene, hg-Tyrs-J, which includes a 1.0-kb 5' flanking sequence of the human tyrosinase gene fused with mouse tyrosinase cDNA. By introducing the fused gene into fertilized eggs of albino mice, we obtained two mice that exhibited pigmentation in the skin and eyes and established a transgenic line from one of them. Further analyses revealed that the transgene was expressed cell type-specifically in these transgenic mice. We conclude, therefore, that the 1.0 kb 5' upstream region of the human tyrosinase gene contains conserved cis-elements essential for cell type-specific expression of the tyrosinase genes in mice and humans. Results of our study may provide a clue to elucidate the evolutionary process of regulatory mechanisms of the tyrosinase gene.

Animals↗
PubMed1979

Chymotrypsin activation of melanosome tyrosinase in hamster melanotic melanoma.

The tyrosinase activity in two sucrose gradient isolated melanosome fractions from a melanotic hamster melanoma was found to increase after alpha-chymotrypsin treatment. The enhancement in tyrosinase activity had its maximum at a concentration of 1 mg/ml alpha-chymotrypsin after 120 min incubation at 37 degrees C. No direct activating effect of alpha-chymotrypsin was found either on the soluble tyrosinase fraction from freshly prepared untreaed whole-tumor homogenate or on purified mushroom tyrosinase. The activating effect of alpha-chymotrypsin upon the melanosome tyrosinase is believed to be due to the endopeptidic hydrolysis of the--CO--NH--bound existing between tyrosinase and tyrosine and phenylalanine residues in the melanin molecule. Although alternative interpretations are not excluded, the observed enhancement in tyrosinase activity after alpha-chymotrypsin treatment of melanosomes might indicate the existence of an "enzyme liberating" mechanism in the melanosomes.

Animals↗
PubMed1975

Tyrosinase as glycoprotein.

Purified tyrosinase T1 was incubated with neuraminidase. The catalytic activity of tyrosinase was essentially retained, after this treatment. The tyrosinase band (Dopa stained) was transformed into a new less anodic form, similar to tyrosinase T2, on disc electrophoresis. The band of protein was also converted to the same position as the Dopa stained. The other hand, the only one PAS stained band of native tyrosinase T1 was splitted into the three slower-moving bands. One was consistent with Dopa and protein stained bands. The other two were much more slower than the former band and completely free of peptide and enzymic activity. The PAS-densitometric value of native tyrosinase T1 was almost equal to those of three separated bands in total. These results suggest that mammalian tyrosinase is a kind of glycoprotein.

Animals↗
PubMed1992

Phylogeny of regulatory regions of vertebrate tyrosinase genes.

Highly homologous DNA elements were found to be shared by the upstream regions of the mouse tyrosinase and tyrosinase related protein (TRP-1) genes. Several nuclear proteins were shown to bind to both of these upstream regions. Shared homologous DNA elements were also found in the 5' flanking sequences of Japanese quail and snapping turtle tyrosinase genes. Shared homologous nucleotide sequences were found to be scattered like an archipelago in the 5' upstream regions of mouse and human tyrosinase genes. Comparisons between Japanese quail and snapping turtle tyrosinase genes gave similar results. On the contrary, mammalian (mouse and human) and nonmammalian (quail and snapping turtle) tyrosinase genes did not show significant homology in their 5' upstream regions. In contrast, coding sequences in the first exons of vertebrate tyrosinase genes and their deduced amino acid sequences were found to be highly conserved except for their putative leader sequence-coding regions.

Amino Acid Sequence↗
PubMed1975

Use of affinity chromatography for purification of tyrosinase.

The possibility of using affinity chromatography for the purification of tyrosinase was explored. When purified mushroom tyrosinase was employed, almost all tyrosinase became bound to Sepharose containing 3-iodotyrosine, an inhibitor of tyrosinase. When crude extracts from B16 melanoma were employed, partial purification of tyrosinase and separation of two or possibly three forms of tyrosinase could be obtained. The results indicate that affinity chromatography could possibly be employed for the purification of tyrosinase during the final stages of the purification procedures.

Basidiomycota↗
PubMed1977

Tyrosinase activity in the sera of patients with malignant melanoma: method and specificity.

Serum tyrosinase activity has been measured by adapting the [3]tyrosine assay for tyrosinase and significant elevations of serum tyrosinase activity were found in patients with malignant melanoma. In contrast to findings in a study which utilized [14C]tyrosine, augmented levels of tyrosinase activity were not observed in sera from patients with other malignancies, including subjects with carcinoma of the breast. The results of the examinations for soluble tyrosinase activity in human malignant melanoma tissue-cultured lines were all positive, whereas human cell lines from carcinoma of the breast, carcinoma of the colon and sarcoma uniformly showed no activity. The method employed for detecting tyrosinase activity holds promise as a specific diagnostic test and may be valuable for monitoring the response to clinical treatment of patients with malignant melanoma.

Breast Neoplasms↗
PubMed1978

The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells.

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Molecular characterization of the mouse tyrosinase gene: pigment cell-specific expression in transgenic mice.

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type-specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.

Albinism↗
PubMed1978

Nature of tyrosinase inactivation in melanosomes.

The decrease in tyrosinase activity following incubation of melanosomes isolated from mouse melanoma with dopa appeared to be related to the degree of in vitro melanization of melanosomes caused by incubation with dopa. The inactivation similarly occurred in the dopa-tyrosinase reaction system containing ascorbic aicd, although in the presence of ascorbic acid the dopa-quinone is immediately converted back to dopa and therefore melanin formation does not take place. The mode of inactivation of tyrosinase in melanosomes appeared to be similar to the reaction inactivation which is known to occur in the reaction of plant plant tyrosinase. Superoxide anion (O-2) and singlet oxygen (1O2) were estimated but undetectable during the dopa-tyrosinase reaction. Thus the inactivation of tyrosinase is not caused by the reaction products or the oxygen radicals.

Animals↗
PubMed1979

Serum tyrosinase in malignant disease, its activity, and the electrophoretic patterns of the enzyme as carried by immunoglobulins'.

Serum tyrosinase activity in many persons with metastatic diseases was found to be significantly higher than activity in normal persons. The highest activity was observed in melanoma and breast carcinoma. The electrophoretic patterns of serum tyrosinase, resolved by electrophoresis of a serum tyrosinase fraction followed by incubation of the gel sample with L-dopa, and represented as sets of RF's of melanin bands, were characteristically different in melanoma, breast carcinoma, and certain other diseases. The RF's of melanin and protein bands in the serum enzyme preparations from melanoma patients were concisely defined. Further, some potent serum fractions inhibiting tyrosinase melanogenic activity have been obtained, and the presence of tyrosinase inhibitors in the serum enzyme preparation has also been demonstrated. More detailed exploration of these serum tyrosinase parameters may provide more specific and sensitive detection for certain malignant diseases.

Breast Neoplasms↗
PubMed1978

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.

Basidiomycota↗