Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new technique based on liquid phase ion exchange chromatography on short column is proposed for quantitative determination of tyrosine and Dopa. alpha-Amino,beta-guanidinopropionic acid is used as an internal standard of coloration. The role of H2O2 and ascorbic acid on tyrosine and Dopa was checked. Ascorbic acid prevents the auto-oxidation of Dopa, H2O2 has no effect on tyrosine but oxidizes Dopa even in the presence of excess ascorbic acid. This method was tested in mushroom tyrosinase, with and without ascorbic acid. Assays performed with tyrosinase from rabbit ocular extracts clearly showed that they do oxidize tyrosine. Reliability of the method is comparable to radioassay.
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PMID:A rapid quantitative analysis of tyrosine and its oxidation products by tyrosinase. 11 92

We have reported previously that 2-hydroxyestradiol can be oxidized in the presence of catechol by mushroom tyrosinase, with a stoichiometric requirement of molecular oxygen (Jacobsohn, G.M. and Jacobsohn, M.K. (1984) Arch. Biochem. Biophys. 232, 189-196). It is then incorporated into melanin (Jacobsohn et al. (1988) J. Steroid Biochem. 31, 377-385). We now report on the isolation and characterization of the o-quinone as a product of the enzyme reaction from 2-hydroxyestradiol. The o-quinone was isolated from incubates and identified by its FTIR spectrum, in particular, by the appearance of a new band at 1652 cm-1, its migration in HPLC systems, its ultraviolet spectrum, its derivatization with phenylenediamine and comparison of these properties with the periodate oxidation product of the same substrate. The enzyme oxidation of the catechol estrogen was performed at 37 degrees C and did not require an activator; dopa at concentrations higher than 5 microM was inhibitory. At concentrations lower than 5 microM, dopa acted catalytically and was not consumed during the course of reaction. Ascorbic acid inhibited the reaction. The quinone exhibited both reversible and irreversible binding to performed melanin and to melanin actively synthesized by the enzyme. Incubation of 18 microM newly synthesized [4-14C]estradiol-2,3- quinone with mushroom tyrosinase for 45 min at 37 degrees C in presence of 400 microM dopa showed incorporation (irreversible binding) of 6.3 +/- 0.3% of label into melanin produced during the course of reaction. Similar incubations for 45 min of pre-formed melanin prepared from 400 microM dopa showed incorporation of 4.4 +/- 0.2% of the label. Reversible binding was 10-times greater than incorporation for both actively synthesized and preformed melanins. In the absence of dopa or catechol, enzyme incubations of either 2-hydroxy-estradiol or its quinone did not yield melanin. Data suggest that estradiol-2,3-quinone is an intermediate in the incorporation of the catechol estrogen into melanin by tyrosinase.
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PMID:Isolation of estradiol-2,3-quinone and its intermediary role in melanin formation. 189 39

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.
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PMID:Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots. 198 92

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.
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PMID:Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma. 309 4

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.
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PMID:pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications. 311 52

The mutagenic activity of quercetin for Salmonella typhimurium TA98 was inhibited by addition of metal salts. MnCl2 was a potent inhibitor, followed by CuCl2, FeSO4, and FeCl3, the probable mechanism being facilitated catalytic oxidation of quercetin. With quercetin incorporated at a level of 100 nmoles/plate, approximate doses (nmoles/plate) to give 50% inhibition of mutagenic activity were: MnCl2 less than 10 (-S9), 18 (+S9); CuCl2 65 (-S9), greater than 100 (+S9); FeSO4 190 (-S9), greater than 300 (+S9); or FeCl3 275 (-S9), greater than 300 (+S9). Ascorbate, superoxide dismutase, and, to a lesser extent, NADH and NADPH, all enhanced the mutagenic activity of quercetin in the absence of the mammalian-microsome (S9) system, but had no significant effect in the presence of the S9 mix. The maximum enhancement of activity by ascorbate or superoxide dismutase was approximately 87% of the increase achieved by addition of the S9 mix. Tyrosinase (catechol oxidase) substantially reduced the mutagenic activity of quercetin in the absence of the S9 mix. At lower levels of tyrosinase, activity was restored by incorporation of the S9 mix. It is proposed that the S9 mix enhances the mutagenic activity of quercetin by scavenging superoxide radicals, thus inhibiting the autoxidation of quercetin, and possibly by reducing quinone oxidation products of quercetin. The mutagenic activity of quercetin increased substantially when the pH of the media was decreased. This may be due in part to a decrease in ionization of quercetin at lower pH, thereby increasing its absorption by the tester strain, to a decrease in the rate of autoxidation of quercetin at lower pH, or to a combination of these.
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PMID:Factors affecting the mutagenic activity of quercetin for Salmonella typhimurium TA98: metal ions, antioxidants and pH. 391 57

The present study was conducted to clarify the mechanism responsible for enhancement of the anti-melanoma activity of levodopa methylester by supplemental ascorbate in vivo. 5-Hydroxydopa, a known cytotoxic agent and the major metabolite formed from levodopa in the presence of ascorbate and mushroom tyrosinase in vitro, was assessed for its antitumor activity against i.p. and s.c. inoculated B16 melanoma, P388 leukemia, and L1210 leukemia in mice with and without supplemental ascorbate. Treatment with 5-hydroxydopa failed to significantly increase survival of mice bearing i.p. or s.c. pigmented and non-pigmented B16 melanomas even though it inhibited local tumor growth. Treatment increased survival of both P388 and L1210 leukemias, and this increase was more pronounced in mice bearing i.p. tumors than in mice bearing s.c. tumors. This treatment significantly decreased final tumor weight of both leukemias implanted s.c., and inhibited ascites formation in mice inoculated with i.p. tumors. Ascorbate supplementation decreased or abrogated the effect of 5-hydroxydopa on survival in mice bearing i.p. or s.c. leukemia tumors and decreased survival relative to control mice bearing i.p. or s.c. pigmented and s.c. non-pigmented tumors. It did not affect survival of treated mice bearing i.p. non-pigmented melanoma tumors. Ascorbate supplementation did not modify the effect of 5-hydroxydopa treatment on primary s.c. tumor growth in mice bearing melanoma or leukemia tumors nor did it affect ascites formation in treated mice bearing i.p. leukemia tumors. The lack of correlation between the observed inhibition of primary tumor growth and the absence of an effect on survival in 5-hydroxydopa treated mice bearing i.p. melanoma may relate to an inability of this drug to interfere with tumor metastasis. These data argue against a role for 5-hydroxydopa as a metabolically derived cytotoxic formed in situ during concurrent treatment with levodopa methylester and supplemental ascorbate.
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PMID:Influence of supplemental ascorbate on the antitumor activity of 5-hydroxydopa, a purported cytotoxic metabolite. 393 10

1. An enzyme from the leaves of spinach beet (Beta vulgaris L.) that catalyses the hydroxylation of p-coumaric acid to caffeic acid in the presence of ascorbate has been purified about 1000-fold on a protein basis. 2. It is activated by high concentrations of ammonium sulphate and sodium chloride. 3. The preparation shows both hydroxylase and catechol oxidase activities, in a constant ratio throughout the purification procedure; they are similarly activated by salts. 4. Ascorbate acts as a reductant in quantities equivalent to the caffeic acid produced by hydroxylation. 5. Ascorbate can be replaced by tetrahydrofolic acid, NADH, NADPH or 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not by caffeic acid. Among these, the pteridine is the most effective, but the reaction is not inhibited by aminopterin. In experiments with saturating concentrations of NADH and the pteridine, these reductants compete in the reaction and are equivalent on a molar basis. 6. No cofactor has been separated from the enzyme by prolonged dialysis. 7. The relation of the enzyme to other hydroxylases and phenolases is discussed.
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PMID:The hydroxylation of p-coumaric acid by an enzyme from leaves of spinach beet (Beta vulgaris L.). 438 84

Reducing agents had no effect on the oxidation of 3,4-dihydroxyphenylalanine (DOPA) to quinone by Mycobacterium leprae; no quinone formation by o-diphenoloxidase of mammalian or plant origin was detected under similar experimental conditions. Ascorbic acid and reduced glutathione prevented further oxidation and polymerization of the quinone to melanin by M. leprae; cysteine was less effective. In the presence of reducing agents, the quinone (indole-5,6-quinone) formed from DOPA by M. leprae was not reduced back to diphenol. On the other hand, the quinone (dopachrome) produced from DOPA by mammalian or plant phenolase was rapidly decolorized by reducing agents. Oxidized glutathione and cystine had little effect on o-diphenoloxidase from all of the three sources. Cyanide, which completely inhibited mammalian and plant phenolases, had only a partial effect on the enzyme in the bacilli. Various lines of evidence suggest that the properties of o-diphenoloxidase in M. leprae are different from those of similar enzymes obtained from other sources.
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PMID:Unusual effects of reducing agents on 0-diphenoloxidase of Mycobacterium leprae. 499 15

1. Partially purified preparations of tobacco-leaf o-diphenol oxidase (o-quinol-oxygen oxidoreductase; EC 1.10.3.1) oxidize chlorogenic acid to brown products, absorbing, on average, 1.6atoms of oxygen/mol. oxidized, and evolving a little carbon dioxide. 2. The effect of benzenesulphinic acid on the oxidation suggests that the first stage is the formation of a quinone; the solution does not go brown, oxygen uptake is restricted to 1 atom/mol. oxidized, and a compound is produced whose composition corresponds to that of a sulphone of the quinone derived from chlorogenic acid. 3. Several other compounds that react with quinones affect the oxidation of chlorogenic acid. The colour of the products formed and the oxygen absorbed in their formation suggest that the quinone formed in the oxidation reacts with these compounds in the same way as do simpler quinones. 4. Some compounds that are often used to prevent the oxidation of polyphenols were tested to see if they act by inhibiting o-diphenol oxidase, by reacting with quinone intermediates, or both. 5. Ascorbate inhibits the enzyme and also reduces the quinone. 6. Potassium ethyl xanthate, diethyldithiocarbamate and cysteine inhibit the enzyme to different extents, and also react with the quinone. The nature of the reaction depends on the relative concentrations of inhibitor and chlorogenic acid. Excess of inhibitor prevents the solution from turning brown and restricts oxygen uptake to 1 atom/mol. of chlorogenic acid oxidized; smaller amounts do not prevent browning and slightly increase oxygen uptake. 7. 2-Mercaptobenzothiazole inhibits the enzyme, and also probably reacts with the quinone; inhibited enzyme is reactivated as if the inhibitor is removed as traces of quinone are produced. 8. Thioglycollate and polyvinylpyrrolidone inhibit the enzyme. Thioglycollate probably reduces the quinone to a small extent.
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PMID:The enzymic oxidation of chlorogenic acid and some reactions of the quinone produced. 594 50


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