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)

Latent mushroom tyrosinase can be considered as a zymogen when activated by proteases because the activation process fulfilled all of the kinetic dependencies predicted by a theoretical zymogen activation model previously reported. The activation was studied under two assay conditions: high and low ratio of latent tyrosinase/serine protease (trypsin and subtilisin Carlsberg) concentrations, in the presence and in the absence of a serine protease inhibitor (aprotinin). The size of the latent enzyme was 67 kDa, determined by denaturing SDS-PAGE electrophoresis and Western blot assays. After proteolytic activation, the size was 43 kDa, with an intermediate band of 58 kDa. The values of the catalytic () and Michaelis () constants for the active forms of tyrosinase resulting from the activation by subtilisin, trypsin, or sodium dodecyl sulfate on the substrate tert-butylcatechol were slightly different, which could support the idea of "one activator-one different active tyrosinase". Vacuum infiltration experiments tried to reproduce in vivo the role of mushroom serine proteases in the activation of latent tyrosinase. The use of serine protease inhibitors is proposed as a new alternative tool to prevent melanin formation.
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PMID:Kinetic study of the activation process of a latent mushroom (Agaricus bisporus) tyrosinase by serine proteases. 1055 77

Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.
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PMID:Characterization of tyrosinase from the cap flesh of portabella mushrooms. 1056 3

Four isoforms of polyphenol oxidase (PPO) were demonstrated in the aerial roots of a tropical orchid, Aranda 'Christine 130'. They were extracted at neutral pH and purified to homogeneity as judged by SDS-gel electrophoresis. Purification was achieved by a combination of Triton X-114 treatment, temperature phase partitioning, gel filtration chromatography, ion-exchange separation and chromatofocusing. Two of the isoforms, designated PPO(a) and PPO(d), differed in their N-terminal sequence, tryptic peptide map and substrate affinity for (+)-catechin, but exhibited similarity in their molecular mass under denaturing conditions, pH optimum and kinetic behaviour toward 4-methyl catechol. The other two isoforms, PPO(b) and PPO(c), were identical to PPO(a) and PPO(d), respectively, in terms of their N-terminal sequence, substrate preference and pH maximum, but were different with regard to their molecular mass under denaturing conditions. These four isoforms differed in their isoelectric point.
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PMID:Characterization of polyphenol oxidase from aerial roots of an orchid, Aranda 'Christine 130' 1058 Feb 84

The enzyme tyrosinase, encoded by tyrosinase gene (mel), is responsible for melanin formation. In a shotgun cloning experiment, a SalI-digested DNA fragment coding for tyrosinase was cloned from Pseudomonas maltophilia DNA into plasmid vector (pUC18) to generate the hybrid plasmid (pWSY). The recombinant plasmid imparted the ability of melanin synthesis to an E. coli host (HB101). The foreign DNA fragment (0.7 kb) possessed no recognition sites for BamHI, HindIII, EcoRI or BclI. Hybridization studies confirmed that the small fragment cloned in pWSY was from P. maltophilia DNA. Nucleotide sequence analysis identified an ORF of 504 nt coding tyrosinase. SDS-PAGE analysis also revealed an additional protein of 18 kDa, which was equal to the putative tyrosinase according to the size of mel fragment, was expressed in the E. coli recombinant carrying the plasmid pWSY.
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PMID:[Cloning and expression of tyrosinase gene from Pseudomonas maltophilia in E. coli]. 1058 69

Melanin biosynthesis is completely inhibited in the B16 melanoma cells following their incubation with inhibitors of the two ER glucosidases. This is primarily due to the inactivation of tyrosinase. Under the same conditions, the DOPA-oxidase activity of TRP-1 was only partially affected. In this report we investigate the effects of the perturbation of N-glycan processing in ER on the transport and activation of tyrosinase and TRP-1. We have localized the DOPA-oxidase activity in normal and inhibited cells and suggest that the first DOPA-reactive compartment of the secretory pathway (trans Golgi network) is also the site of tyrosinase activation. The inhibition of N-glycan processing does not affect the intracellular trafficking of the two melanogenic enzymes that are correctly transported to melanosomes. Immunoprecipitation experiments followed by analysis in SDS-PAGE under non-reducing conditions suggest that in inhibited cells, both tyrosinase and TRP-1 are synthesized in a modified conformation as compared to the normal proteins. These data suggest that the inhibition of melanin synthesis is not due to a defective transport but rather to conformational changes induced in the structure of tyrosinase and TRP-1 during their transit through the ER.
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PMID:Investigation of the intracellular transport of tyrosinase and tyrosinase related protein (TRP)-1. The effect of endoplasmic reticulum (ER)-glucosidases inhibition. 1064 4

Melanin, the phenolic biopolymer that serves as a skin- and hair pigment-protecting agent against harmful solar radiation and a free radical trap, is biosynthesized in animals mainly by the action of tyrosinase also known as phenoloxidase. Regulation of tyrosinase and hence melanogenesis is vital for all animals. In this report, we present the isolation and characterization of a new, heat-labile glycoprotein inhibitor of phenoloxidase from the larvae of Manduca sexta. The inhibitor was isolated from the live larval cuticle by buffer extraction and purified to homogeneity employing ammonium sulfate precipitation, dialysis, and concanavalin A-Sepharose chromatography. It migrated with a molecular weight of 380,000 on SDS-PAGE gels and inhibited the activity of insect and plant as well as fungal phenoloxidases. Inhibitor formed a tight complex with phenoloxidases, which resisted dissociation even by 1% Triton X-100 or SDS. Selective inhibition of phenoloxidase, while acting on certain but not all different substrates, was observed. The physiological importance of this newly discovered high-molecular-weight phenoloxidase inhibitor is discussed.
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PMID:Characterization of a new phenoloxidase inhibitor from the cuticle of Manduca sexta. 1067 12

Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. Gel filtration, N-terminal amino acid sequencing and SDS/PAGE analysis indicate that T. roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da. The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L-3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) and pyrogallol. The K(m) value of the enzyme for L-DOPA was 0.18 mM. beta-Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 degrees C, and was increased by addition of 1 mM Mg(2+), K(+) or Cu(2+). The enzyme was highly stable against high temperature and guanidine hydrochloride. The N-terminal amino acid sequence of the enzyme was determined to be Asp-Ile-Asn-Gly-Gly-Gly-Ala-Thr-Leu-Pro-Gln-Lys-Leu-Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources.
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PMID:Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum. 1074 56

Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (> 300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with different relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens.
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PMID:Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish. 1093 21

Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid.
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PMID:Latent polyphenol oxidases from sago log (Metroxylon sagu): partial purification, activation, and some properties. 1105 75

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.
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PMID:Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants. 1114 Dec 62


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