Secretion of Dopa in Phaeochromocytoma

In a series of 11 cases with phaeochromocytoma, two patients were found in whom the tumour secreted dopa in addition to dopamine, noradrenaline, and adrenaline. These two patients were normotensive in spite of high plasma and urinary levels of noradrenaline and adrenaline. This raises the possibility that dopa may be able to protect against the hypertensive action of noradrenaline. Such a mechanism would also explain the absence of hypertension in many cases of neuroblastoma.     

Accurate assay of dopa decarboxylase by preventing nonenzymatic decarboxylation of dopa

The nonenzymatic decarboxylation of dopa was completely blocked by both 2-mercaptoethanol and EDTA together over the wide range of pH. This finding made it possible to measure the activity of dopa decarboxylase precisely even at an alkaline pH value. The pH optimum of dopa decarboxylase was found to be pH 7.0 and the Km value for dopa was determined to be 4 X 10(-5) M.     

Human Melanoma Cell Lines Show Little Relationship Between Expression of Pigmentation Genes and Pigmentary Behaviour In Vitro

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with α-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by α-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; α-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or α-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinaserelated genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.     

Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation.

1. Melanosomal tyrosinase was isolated from normal C57B1 mice, and a comparison of the tyrosine-hydroxylation and dopa (3,4-dihydroxyphenylalanine)-oxidation activities of this enzyme was made. 2. The results indicate that in the absence of dopa cofactor, this enzyme is capable of tyrosine hydroxylation, but with very little subsequent dopa oxidation and melanin formation. 3. This mechanism of enzyme action may play an important role in the intracellular regulation of melanin formation. 4. Further, dopa appears to act as a positive allosteric effector for tyrosine hydroxylation by tyrosinase, in addition to its known activity as a hydrogen donor for the reaction.     

Chiasma-induction and tyrosine metabolism in locusts

The production of a gregarization pheromone has been postulated in locusts, with effects on melanization of the hopper cuticle and increased chiasma frequency during meiosis in the adult on crowding or gregarization. Lack of chiasma-inducing effect of the pheromone on albino strains is correlated with the absence or deficiency of some of the products of the metabolic pathways of tyrosine. Some of these products, commercially obtainable, are the amino acids phenylalanine and tyrosine leading to both the melanization and sclerotization pathways; dopamine formed from dopa in the lastnamed pathway; three products of dopamine i.e. protocatechuic acid, noradrenaline and adrenaline. The injection of solutions of these metabolites into the haemolymph of solitary hoppers has shown that only dopa to some extent but noradrenaline to a large extent are effective in raising chiasma frequency in solitarised individuals of normal-coloured strains of Locusta, while in two albino strains, which differ genetically, the injection of dopa, dopamine, protocatechuic acid and noradrenaline proved effective; phenylalanine was effective in only one of these albino strains, while adrenaline was effective in neither. The chiasma-inducing effect of noradrenaline, common to the three strains, is accompanied in the normal-coloured strain by a greater retention of dark coloration during solitarization and by some attainment of the crowded type of morphometric ratios which is a third physical criterion of gregarization. The genetic blocks to the physical criteria of gregaria in the albino strains lie at the immediate level of dopa production or previous to this reaction; it may be construed that such a block in the solitaria of normal-coloured strains also lies at this early level, in this case being induced by too low a pheromone concentration.     

A kinetic analysis of Drosophila melanogaster dopa decarboxylase

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.     

Indirect oxidation of 6-tetrahydrobiopterin by tyrosinase

6-Tetrahydrobiopterin is known to bind to an allosteric site of tyrosinase to directly inhibit the enzyme. However, simultaneous measurements of ultraviolet-visible absorption spectra and oxygen consumption led us to conclude that the inhibition was due to oxidation of 6-tetrahydrobiopterin by dopaquinone. Immediately after addition of 6-tetrahydrobiopterin, tyrosinase stopped producing dopachrome from either tyrosine or dopa. Duration of inhibition was proportional to the concentration of added 6-tetrahydrobiopterin and the enzyme activity was fully restored after the inhibition. Surprisingly, there was a rapid consumption of oxygen during the inhibition period. In addition, absorption spectra indicated that the only reaction that occurred during the inhibition was oxidation of 6-tetrahydrobiopterin to 7,8-dihydrobiopterin. In the absence of tyrosine or dopa, tyrosinase did not oxidize 6-tetrahydrobiopterin, suggesting that a reaction intermediate between dopa and dopachrome was a target for the inhibition. We propose a new mechanism in which dopa is oxidized to dopaquinone and the latter, instead of producing dopachrome, is reduced back to dopa by 6-tetrahydrobiopterin.     

The effect of oxygen on melanin precursors released from retinal pigment epithelial cells in vitro.

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240-275 nm and a maximum around 300 nm with a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

The Effect of Oxygen on Melanin Precursors Released from Retinal Pigment Epithelial Cells In Vitro

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240–275 nm and a maximum around 300 nm wth a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

Phosphorylated mixed isomers of L-dopa increase melanin content in skins of Skh-2 pigmented hairless mice

Dopa phosphates, a new class of compounds, contain phosphate-ester linkages at the 3- and/or 4- positions of the phenylalanine ring of L-dopa. Dopa phosphates have been shown to increase pigment production in the epidermis of hairless mice. Groups of Skh-2 pigmented hairless mice were treated topically with various concentrations of dopa phosphates daily for five weeks. Half of each group received suberythemal UVB radiation three times weekly for four weeks from a bank of filtered FS20 lamps. UVB and dopa phosphates alone each caused a modest increase in epidermal pigmentation. However, treatment of mice with dopa phosphates plus UVB radiation resulted in a marked increase in pigmentation, greater than with either treatment alone. The optimal concentration of dopa phosphates was 0.01% (100 micrograms/ml Tris-glycerol buffer) whether or not they were applied in conjunction with UVB radiation. Histological analyses revealed that dopa phosphates and UVB radiation each caused an increase in the number of pigmented melanocytes in the epidermis. Control groups treated with Tris-glycerol buffer alone, or buffer containing L-phenylalanine or L-dopa showed no significant changes in pigmentation. Our results indicate that dopa phosphates stimulate the production of melanin and affect the development and distribution of melanocytes in the skin of Skh-2 mice. By these criteria, dopa phosphates and UVB act in a similar manner to increase melanin content in the skin. The processes may be related to those recently observed in cultured mouse melanoma cells where dopa phosphates are incorporated into melanin, presumably following enzymatic hydrolysis by cellular phosphatases with the resultant production of L-dopa and inorganic phosphate.     

Mussel protein adhesion depends on interprotein thiol-mediated redox modulation

Mussel adhesion is mediated by foot proteins (mfps) rich in a catecholic amino acid, 3,4-dihydroxyphenylalanine (dopa), capable of forming strong bidentate interactions with a variety of surfaces. A tendency toward facile auto-oxidation, however, often renders dopa unreliable for adhesion. We demonstrate that mussels limit dopa oxidation during adhesive plaque formation by imposing an acidic, reducing regime based on the thiol-rich mfp-6, which restores dopa by coupling the oxidation of thiols to dopaquinone reduction.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

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 tyrosinase, 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.     

Hydroxycinnamic acid esters enhance peroxidase-dependent oxidation of 3, 4-dihydroxyphenylalanine. differences in the enhancement among the esters

Horseradish peroxidase (HRP)-dependent oxidation of 3, 4-dihydroxyphenylalanine (dopa) was studied to elucidate the mechanism of its oxidation. The oxidation of dopa was enhanced by hydroxycinnamic acid esters and dopa supressed HRP-dependent oxidation of the esters. These results indicate that phenoxyl radicals of hydroxycinnamic acid esters that are formed at first, can oxidize dopa. Among hydroxycinnamic acid esters used, affinity of the phenoxyl radicals for dopa was in order 4-coumaric>caffeic>ferulic acid ester radicals.     

A study of the supposed hydroxylation of tyrosine catalysed by peroxidase.

The claim that peroxidase (rather than tyrosinase) is the enzyme responsible for the conversion of tyrosine into dopa (3,4-dihydroxyphenylalanine) in melanogenesis was investigated. The spectral changes that occurred during the action of horseradish peroxidase in the presence of H2O2 on dopa, tyrosine and mixtures of dopa with tyrosine or other phenolic compounds were studied. The effect of ascorbic acid or dihydroxyfumaric acid on some of these changes was also investigated. No evidence was found that tyrosine was hydroxylated by peroxidase in the presence of H2O2 and dopa as cofactor, although tyrosine or other phenolic compounds increased the rate of oxidation of dopa to dopachrome (indoline-5,6-quinone-2-carboxylic acid). Peroxidase was, however, effective in oxidizing tyrosine to dopa in the presence of dihydroxyfumaric acid and oxygen.     

Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

Developmental regulation of catecholamine levels during sea urchin embryo morphogenesis

Results of a number of pharmacological studies suggest that catecholamines play a regulatory role in cleavage, morphogenesis and cell differentiation during early animal embryonic development. Few studies, however, have actually assayed for levels of catecholamines in these early embryos by methods that are both sensitive and specific. In this investigation the catecholamines dopamine, norepinephrine and epinephrine and their precursor, dopa and metabolites were determined in eight different embryonic stages of the sea urchin, Lytechinus pictus from hatched blastula to late pluteus larva, using high performance liquid chromatography with electrochemical detection. Levels of each of the catecholamines exhibited unique developmental profiles and are consistent with a role for epinephrine in blastula and early gastrula embryos and for norepinephrine in gastrulation. Changes in levels of catecholamine precursor and metabolites suggest a changing pattern of synthetic and metabolic enzyme activity, which can, for the most part, explain the fluctuations in catecholamine levels during development from blastula to the pluteus larva stage.     

Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase

Free tyrosine and tyrosine residues in various peptides and proteins are converted into dopa and dopa residues by tyrosinase (monophenol,L-dopa:oxygen oxidoreductase, EC 1.14.18.1) in the presence of reductants. The efficiency of the tyrosine-to-dopa conversion was examined under varied conditions, such as the substrate-to-tyrosine ratio, concentrations of reductant and oxygen in the reaction solution, pH, temperature and reaction time. The highest dopa yields were achieved with the following optimal conditions for hydroxylation: 0.1 M phosphate buffer at pH 7, 25 mM ascorbic acid, 1 mM tyrosine, 50 micrograms/ml tyrosinase and 20 degrees C. Using these conditions, up to 70% of free tyrosine was converted into dopa, and tyrosine residues in several synthetic peptides were also hydroxylated to dopa residues at ratios as high as free tyrosine. The preparation of hydroxylated analogues of the decapeptide (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys), in particular, may contribute to a better understanding of adhesion in the dopa-containing mussel glue protein.