Biomedical subjects

A Wittbjer

Publications and source records attributed to A Wittbjer.

13 recordsLinked to original sources

PubMed1996

Enzymatic and non-enzymatic oxygenation of tyrosine.

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. L-Tyrosine and D-tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of L-tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of D-tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the L-isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with L- and D-tyrosine, human tyrosine, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether D-tyrosine or L-tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.

Animals↗

PubMed1994

Neuromelanin of the human substantia nigra: a mixed-type melanin.

Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO4 and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin-derived compound, thiazole-4,5-dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO4 and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed-type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.

Autopsy↗

PubMed1994

The mouse brown (b) locus protein functions as a dopachrome tautomerase.

The mouse b locus controls black/brown coat coloration. Its product, the b-protein or TRP-1, has significant homology to tyrosinase, and this has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have used lines of mouse fibroblasts stably expressing the b-protein. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b-protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melanin metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b-protein. Culture medium from the line expressing both proteins contains significant amounts of methylated carboxylated indoles, such as 6-hydroxy-5-methoxyindole-2-carboxylic acid, which would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosinase alone are approximately 20-fold lower, and not significantly above background. Hence, it appears that the b-protein acts as a dopachrome tautomerase in vivo as well as in vitro.

3T3 Cells↗

PubMed1993

Fibroblasts expressing mouse c locus tyrosinase produce an authentic enzyme and synthesize phaeomelanin.

Recent advances in the study of the molecular biology of mouse pigmentation have led to the discovery of a family of proteins involved in the control of melanin synthesis. It has been confirmed that the product of the mouse c (albino) locus is the key melanogenic enzyme tyrosinase, but study of its function and regulation have been hampered by the presence of closely related proteins within melanin-synthesising cells. To overcome these problems, we have established lines of mouse fibroblasts expressing the c locus mouse tyrosinase. Here we describe characterisation of the tyrosinase synthesised by these cells and demonstrate considerable similarity between the expressed tyrosinase and the native enzyme. The expressed tyrosinase is proteolytically cleaved to produce membrane-bound and soluble forms of the expected molecular mass and is rich in N-linked carbohydrate, suggesting that melanocytic differentiation is not a prerequisite for post-translational modification of the protein. The expressed enzyme has tyrosinase activity, but not catalase or dopachrome tautomerase activity, confirming that it is an authentic tyrosinase. Transfected fibroblasts expressing tyrosinase are shown to share several physiological characteristics with melanoma cell lines, including increased pigmentation and tyrosinase activity in response to increased cell density. Since tyrosinase is expressed under a heterologous promoter, these shared characteristics probably reflect translational or post-translational controls that operate in both non-melanocytic and melanocytic cell types. We demonstrate that pigmented fibroblasts contain the melanin synthesis intermediates 5-S-cysteinyldopa and 5-S-glutathionyl-dopa, and produce a phaeomelanin-like pigment, but do not contain detectable eumelanin. Expression of tyrosine is therefore sufficient for the synthesis of a form of melanin pigment in fibroblasts.

3T3 Cells↗

PubMed1993

The mouse brown (b) locus protein has dopachrome tautomerase activity and is located in lysosomes in transfected fibroblasts.

Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers beta-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.

3T3 Cells↗

PubMed1993

Hydrogen peroxide as an inducer of elevated tyrosinase level in melanoma cells.

The effects of systems generating active oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) on tyrosinase have been studied in cultured human melanoma cells. Tyrosinase activity was determined by measuring the quantity of 5-S-L-cysteinyl-L-dopa (5-S-CD) formed in the presence of D,L-dopa and L-cysteine. In some experiments, the enzyme protein was determined by radio immunoassay [RIA]. Exposure of cells to xanthine/xanthine oxidase or glucose/glucose oxidase resulted in a dose-related elevation of tyrosinase. Catalase, but not superoxide dismutase, prevented this increase indicating that hydrogen peroxide may be the agent responsible for the action, whereas superoxide anion is not involved. Hydroxyl radicals formed by the Haber-Weiss or Fenton type reactions were not found to produce elevation of tyrosinase. Catalase determinations showed no enzyme in the medium but a high concentration in the cells. Inhibition of intracellular catalase by 3-amino-1,2,4-triazole caused an increase in the tyrosinase level. The effects of dopac, xanthine/xanthine oxidase, and glucose/glucose oxidase all producing hydrogen peroxide, and increasing tyrosinase, were enhanced by the inhibition of catalase. It is concluded that hydrogen peroxide, formed by the systems, accounts for the elevation of tyrosinase level. When tyrosinase activities determined by 5-S-CD formation were compared to enzyme amounts found by RIA, the ratios of these values were always constant. This fact indicates that the increase in the tyrosinase activities was not due to an activation of the enzyme, but mirrored the quantities of enzyme protein present in the samples. On the basis of our findings, it is assumed that hydrogen peroxide is a regulator of tyrosinase in normal melanocytes and melanoma cells.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1991

A sensitive tyrosinase method for human skin.

A sensitive competitive radioimmunoassay for quantitative analysis of tyrosinase in biological tissues has been developed. The binding sites of antityrosinase antibodies were competed for by 125I-labelled human tyrosinase and a known (standard) or unknown (sample) amount of tyrosinase in solution. A sensitivity range of about 5-500 pmol/l was obtained when a solubilized melanoma tyrosinase with a molecular weight of 66,000 D was used as a standard. The method was used on 0.5 cm2 skin samples from which tyrosinase was solubilized with Nonidet P-40. The method is useful for measuring the content of tyrosinase in crude samples where the enzyme activity may be affected by inhibitors or by unspecific oxidation.

Animals↗

PubMed1990

Isolation of tyrosinase from bovine eyes.

Pigmented tissues from bovine eye were used as a source for isolation of tyrosinase from normal melanocytes. Tyrosinase is highly hydrophobic and the isolation procedure is mainly based on the use of hydrophobic interaction chromatography. The bovine enzyme is, in contrast to the human melanoma tyrosinase, mainly soluble. The predominant part of the ocular enzyme from cow has a molecular weight and isoelectric behavior similar to that of the soluble tyrosinase in the human melanoma cells. The N-terminal amino acid sequence of isolated bovine tyrosinase was determined by automated Edman degradation. The N-terminal amino acid sequence from normal bovine tyrosinase was identical to the sequence of an N-terminal region of mouse melanoma tyrosinase predicted from a c-DNA clone by Kwon et al. (1988). The amino acid sequence of bovine tyrosinase shows homology to that of human tyrosinase (Wittbjer et al., 1989), but three amino acids of the 16 residues determined by us differed. Histidine was the N-terminal amino acid.

Amino Acid Sequence↗

PubMed1990

Isolation of soluble tyrosinase from human melanoma cells.

In the human melanoma cell tyrosinase exists in a membraneous and a soluble form. The membraneous enzyme has an N-terminal amino acid sequence identical to that predicted from a human c-DNA clone by Kwon et al.. The soluble form has now been isolated by a technique mainly based on the trypsin resistence of the enzyme and the use of hydrophobic interaction chromatography. The specific dopa oxidase activity of the soluble enzyme was 300 mumol/min x mg protein. On isoelectric focusing the enzyme was found in at least ten bands, pI between 3.8-4.6. The molecular weight was found to be 53,000 D. The N-terminal amino acid sequence was the same as that found in the membrane bound form of the enzyme, i.e. the protein maps at the c-albino locus.

Amino Acid Sequence↗

PubMed1989

Tyrosinase activity in serum from patients with malignant melanoma.

A preparation procedure is presented for the determination of tyrosinase-catalysed stereo-specific dopa oxidase activity in serum. Purification is obtained by separation on a Phenyl-Sepharose hydrophobic interaction column, followed by Con-A-Sepharose chromatography. Five out of seven sera from patients with widespread melanoma metastases were found to contain detectable quantities of tyrosinase. There was no tyrosinase activity in seven sera from patients with other malignancies, nor in six other control sera from individuals without malignancies. One serum which showed high tyrosinase activity was processed as above and studied by SDS-PAGE. A dopa-reactive band with an apparent MW of 66 kD was present in the gel, i.e. at the same place as that of the soluble tyrosinase of cultured human malignant melanoma cells. The protein was found to have the same pI at isoelectric focusing, and eluted in the same way from the preparation columns used, as did soluble tyrosinase.

Aged↗

PubMed1989

Isolation of human tyrosinase from cultured melanoma cells.

Tyrosinase was isolated from cultured melanoma cells using a procedure involving solubilization of the enzyme by means of Triton X-100, followed by different types of chromatography and tryptic digestion to make the enzyme soluble even in the absence of detergent. Starting with a membranous material containing 72 mg protein, 0.21 mg tyrosinase was obtained. The recovery of tyrosinase was 36% of the quantity found in the membranous starting material. In order to acquire a completely purified enzyme preparation suitable for amino acid sequence analysis, SDS-PAGE followed by blotting onto a polyvinylidene difluoride membrane was performed as a final step. The apparent molecular weight was found to be 66,000. Determination of the amino acids of the aminoterminal portion by automated Edman degradation showed the following sequence: His-Phe-Pro-Arg-Ala-X-Val-Ser-Ser-Lys-Asn-Leu-Met-Glu-Lys-Glu-X-X-Pro-Pr o-The enzyme purified has an amino acid sequence identical with that of human tyrosinase deduced from c-DNA by Kwon et al. Striking similarities between our amino acid sequence and that predicted by Yamamoto et al. from mouse tyrosinase c-DNA were also observed.

Amino Acid Sequence↗

PubMed1985

Production of 6-hydroxydopa by human tyrosinase.

A tyrosinase obtained from cultured human melanoma cells was found to oxygenate 2,4-dihydroxyphenylalanine to the strongly cytotoxic amino acid 6-hydroxydopa (2,4,5-trihydroxyphenylalanine). The oxygenation was dependent on the presence of a reducing co-substrate such as dopa or dopamine. The rate of oxygenation of 2,4-dihydroxyphenyl-D,L-alanine was similar to that of L-tyrosine, the normal substrate of tyrosinase. The enzymatic reaction demonstrated may prove of value in the chemotherapy of human melanoma.

Catalysis↗

PubMedDate not supplied

Melanin-related biochemistry of IGR 1 human melanoma cells.

Cultured melanoma cells have been of great value in the study of pigment metabolism. IGR 1 human melanoma cells, established by Dr Christian Aubert, produce melanin in large quantities. These cells have been used for isolation of human tyrosinase which enzyme has not previously been obtained in a pure form. IGR 1 cells contain large amounts of 5-S-cysteinyldopa which is the quantitatively most important catecholic amino acid. This review deals with the metabolism of dopa, cysteinyldopa, glutathionyldopa, cysteine and glutathione, compounds of central importance in pigment metabolism. The information available on tyrosinase, catecholic compounds and on thiols in IGR 1 melanoma cells makes these cells most suitable for further investigation of the metabolism of human melanoma cells.

3,4-Dihydroxyphenylacetic Acid↗