Mammalin tyrosinase. Stoichiometry and measurement of reaction products

Hearing, Vincent J.; Ekel, Thomas M.; Montague, Paul; Nicholson, Jesse M.

doi:10.1016/0005-2744(80)90061-3

Cited by 81 publications

(43 citation statements)

References 23 publications

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“…Thus, crayfish proPO has a substrate specificity that is similar to other tyrosinases. The substrate specificity of the crustacean phenoloxidase is very similar to insect tyrosinases, whereas vertebrate tyrosinases have a more limited substrate specificity (38)(39)(40). In addition, inhibitors that typically inhibit tyrosinases, such as diethyldithiocarbamate, phenylthiourea, and 4-nitrocatechol, were effective in inhibiting the L-dihydroxyphenylalanine-oxidizing activity of the crayfish proPO (Table 1).…”

Section: Fginshhwhwhlvypiemnvn-----rdr------------------kgelfyymhqqmvmentioning

confidence: 99%

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

Aspán

Huang

Cerenius

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

220

137

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Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition system in crustaceans and insects, has been purified and cloned from a crayfish blood cell cDNA library. The deduced amino acid sequence codes for a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, the apparent mass of the purified enzyme. proPO contains two copper atoms, and two putative copper-binding sites were found in the deduced amino acid sequence. Sequence comparisons show that these putative copper-binding sites are similar to the corresponding sites in arthropod hemocyanins and also, although the sequence similarities are less extensive, similar to tyrosinases from vertebrates and microorganisms. The purified enzyme is a typical tyrosinase because it hydroxylates monophenols and oxidizes odiphenols but does not oxidize p-diphenols. If a homogeneous preparation of crayfish proPO were incubated with a homogeneous sample of the proPO activating enzyme, a serine proteinase, the cleavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and

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Section: Fginshhwhwhlvypiemnvn-----rdr------------------kgelfyymhqqmvmentioning

confidence: 99%

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

Aspán

Huang

Cerenius

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

220

137

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“…One milliliter of each FFE fraction was recovered and centrifuged at 14,000 ϫ g for 30 min. The pellets were resuspended in 30 l of extraction buffer [1% Nonidet P-40͞0.01% SDS͞0.1 M Tris⅐HCl, pH 7.2, and a protein inhibitor mixture (Roche Molecular Biochemicals)], vortexed, and kept at 4°C for 1 h. TYR and DCT activities were then measured as described (2,28,29).…”

Section: Preparation Of Sucrose Density Gradient-purified Melanosomesmentioning

confidence: 99%

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

Kushimoto

Basrur

Valencia

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

215

263

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Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.pigment ͉ melanin ͉ tyrosinase ͉ melanoma M ore than 95 distinct genes that play direct or indirect roles in mammalian pigmentation have been identified. Many of these genes encode proteins that are localized in melanosomes, specialized pigment organelles produced only by melanocytes. These gene products alter the quality or quantity of melanin produced and͞or the processing and distribution of melanosomes. The known melanosomal proteins are involved in melanogenesis as catalytic and͞or structural components. These include tyrosinase (TYR), the tyrosinase-related proteins-1 and -2 (TYRP1͞TRP1 and DCT͞TRP2, respectively; refs.

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“…The trapping of label in melanin is unlikely to be a serious problem. Hearing et al have reported that melanin formed from ~-[carhoxy-'~C)tyrosine by mammalian tyrosinase contains only approximately 1% of the I4C initially present in the L-tyrosine substrate, the remainder of the label being released as I4CO2 further along the pathway [31]. The use of D-dopa rather than L-dopa as a cofactor has notable advantages.…”

Section: Tyrosine Lzydroxylase Assuymentioning

confidence: 99%

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase

Winder

Harris

1991

European Journal of Biochemistry

262

219

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New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses ~-[carboxy-'~C]tyrosine as the substrate. I4CO2 is released from the products of the hydroxylation and further metabolism of ~-[carboxy-'~C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.It is well established that tyrosinase from lower organisms catalyses the first two steps of melanin synthesis: the hydroxylation of tyrosine to dopa, and the oxidation of dopa to dopaquinone [l]. The classical view is that this is also true for the mammalian enzyme (21; it has been suggested that mammalian tyrosinase may catalyse a third step in melanin synthesis: the oxidation of 5,6-dihydroxyindole [3, 41. There have, however, been reports of the separation of melanogenic tyrosine hydroxylase and dopa oxidase activities on purification of the mammalian enzyme [5, 61. To determine conclusively whether the tyrosine hydroxylase and dopa oxidase activities of tyrosinase are inherent in one protein molecule, satisfactory assays for these two activities are required. There are many problems associated with the existing assays so the aim of this work was to develop improved assays for both activities. Tyrosine hydroxylase assayThe tyrosine hydroxylase activity of tyrosinase requires Ldopa, the product of the reaction, as a cofactor. In the absence of cofactor, no activity [7] or very low activity [8] has been reported. In general, a cofactor is required at a low concentration relative to the substrate, but a relatively high concentration of L-dopa is required [9]. If tyrosinase does catalyse the first two steps of melanin synthesis, addition of L-dopa as a cofactor also represents addition of a competitive substrate. Oxidation of L-dopa by dopa oxidase activity will deplete the concentration of cofactor. The requirement for L-dopa as a cofactor thus makes the tyrosine hydroxylase activity of tyrosinase difficult to assay.The Pomerantz method [lo] is used most frequently to assay the tyrosine hydroxylase activity of tyrosinase. It uses ~-[3,5-~H]tyrosine as the substrate and measures the 3 H 2 0Correspondence to A.

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Mammalin tyrosinase. Stoichiometry and measurement of reaction products

Cited by 81 publications

References 23 publications

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase

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