Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6‐dihydroxyindole and 5,6‐dihydroxyindole‐2‐carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6‐dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.     

Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.     

Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mm concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med.46, 535–540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000.     

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.     

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.     

The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein

The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure.     

Fluorescence histochemical demonstration of dopa thioethers by condensation with gaseous formaldehyde

Summary The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas glutationedopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480–485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilibrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.     

The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine hydroxylase level in the brain

Tyrosine hydroxylase (TH) is a rate‐limiting enzyme for dopamine synthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. BH4 deficiency leads to the loss of TH protein in the brain, although the underlying mechanism is poorly understood. To give insight into the role of BH4 in the developmental regulation of TH protein level, in this study, we investigated the effects of acute and subchronic administrations of BH4 or dopa on the TH protein content in BH4‐deficient mice lacking sepiapterin reductase. We found that BH4 administration persistently elevated the BH4 and dopamine levels in the brain and fully restored the loss of TH protein caused by the BH4 deficiency in infants. On the other hand, dopa administration less persistently increased the dopamine content and only partially but significantly restored the TH protein level in infant BH4‐deficient mice. We also found that the effects of BH4 or dopa administration on the TH protein content were attenuated in young adulthood. Our data demonstrate that BH4 and catecholamines are required for the post‐natal augmentation of TH protein in the brain, and suggest that BH4 availability in early post‐natal period is critical for the developmental regulation of TH protein level.     

Administration of Dihydroxyphenylalanine to Parkinsonian Patients

Dihydroxyphenylalanine (dopa), the metabolic precursor of dopamine, was administered to 10 parkinsonian patients in oral doses of 1-5 g. and intravenous doses of 0.2-0.5 g. Increases in dopamine excretion of 100- to 1000-fold following the dopa administration indicated that dopa was being absorbed and metabolized. Only two of the 10 patients showed any objective improvement on this treatment. Although dopa did not show sufficient beneficial results in this study to be considered a useful therapeutic agent, its slight activity is consistent with other evidence suggesting that some extrapyramidal cells are sensitive to dopamine.     

The impact of specific oxidized amino acids on protein turnover in J774 cells

Oxidized protein deposition and accumulation have been implicated in the aetiology of a wide variety of age-related pathologies. Protein oxidation in vivo commonly results in the in situ modification of amino acid side chains, generating new oxidized amino acid residues in proteins. We have demonstrated previously that certain oxidized amino acids can be (mis)incorporated into cell proteins in vitro via protein synthesis. In the present study, we show that incorporation of o- and m-tyrosine resulted in increased protein catabolism, whereas dopa incorporation generated proteins that were inefficiently degraded by cells. Incorporation of higher levels of L-dopa into proteins resulted in an increase in the activity of lysosomal cathepsins, increased autofluorescence and the generation of high-molecular-mass SDS-stable complexes, indicative of protein aggregation. These effects were due to proteins containing incorporated L-dopa, since they were not seen with the stereoisomer D-dopa, which enters the cell and generates the same reactive species as L-dopa, but cannot be incorporated into proteins. The present study highlights how the nature of the oxidative modification to the protein can determine the efficiency of its removal from the cell by proteolysis. Protection against the generation of dopa and other species that promote resistance to proteolysis might prove to be critical in preventing toxicity from oxidative stress in pathologies associated with protein deposition, such as atherosclerosis, Alzheimer's disease and Parkinson's disease.     

Hydroxylating activity of frog epidermis tyrosinase.

Trypsin activated in a similar way both the tyrosine hydroxylase and the dopa-oxidasa activities of frog epidermis tyrosinase. Several electron donors reduced or eliminated the lag period for the hydroxylating enzyme. 4 x 10(-5) M dopa was particularly effective, but without affecting the stationary activity after lag period. Tyrosine hydroxylase had KM = 2.6 X 10(-3) M for tyrosine and 2 x 10(-3) M dopa was a competitive inhibitor with Ki = 5 x 10(-4) M. The enzyme was inactivated during its actuation. Data on thermal denaturation were similar to other obtained from dopa oxidase. Our results tend to confirm our previous hypothesis that the activatory process of the enzyme is accompanied by a spatial unfolding of the enzyme molecule.     

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.     

3, 4-Dihydroxyphenylalanine is oxidized by phenoxyl radicals of hydroxycinnamic acid esters in leaves ofVicia faba L

Mechanisms of oxidation of 3,4-dihydroxyphenylalanine (dopa) in leaves ofVicia faba have not yet been elucidated in details. The author hypothesized its oxidation by radicals of hydroxycinnamic acid esters that were generated by a peroxidase-dependent reaction in vacuoles. The results obtained in this study were followings. 1) Vacuolar peroxidase isolated from the leaves oxidized dopa more slowly than 4-coumaric and caffeic acid esters isolated from the leaves. 2) The hydroxycinnamic acid esters enhanced peroxidase-dependent oxidation of dopa and dopa suppressed their oxidation. 3) Degree of the enhancement was roughly correlated with rates of the oxidation of hydroxycinnamic acid esters. 4) The hydroxycinnamic acid esters increased levels of dopa radical in the presence of peroxidase. 5) In protoplasts of mesophyll cells ofV. faba, hydrogen peroxide-induced oxidation of dopa was faster than that of 4-coumaric acid and caffeic acid esters. These results support the above hypothesis that dopa in vacuoles is oxidized by phenoxyl radicals of hydroxycinnamic acid esters that are generated by vacuolar peroxidase.     

The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway

The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.     

Entropy-driven polymerization of ribgrass virus protein

Holmes ribgrass virus (HRV), because of serological results, is regarded as a distantly related strain of tobacco mosaic virus (TMV). HRV protein differs substantially in amino acid sequence from TMV protein, especially in that it contains one histidine residue and three methionine residues, compared to none of either for TMV protein. Ultracentrifugation and hydrogen ion titration data on HRV protein, similar to those obtained previously for the early stage polymerization of TMV and E66 proteins, demonstrated some similarities and more distinct differences from those of the other two proteins. The major similarities are that the early polymerization of HRV protein is entropy driven and the first major polymerized product is a 20 S component, presumably a double disk or two-turn helix, as in the case of the other proteins. The major differences are that the unpolymerized HRV protein sediments at 3 S rather than at the 4 S for the others; it is presumably a dimer of the polypeptide chain. The enthalpy of polymerization per mole of A protein, delta H*, is 18,400 cal for HRV protein, compared to about 30,000 for TMV protein. One mol of H+ ion/mol HRV A protein, compared to 1.5 for TMV and E66 proteins, is bound during polymerization to the 20 S state. Contrasted with the other proteins, very little if any electrical work contribution was detected for the HRV protein. A major difference was found in hydrogen ion titration. Unpolymerized HRV protein binds hydrogen ions significantly in the unpolymerized A protein state, unlike the A proteins from the other two viruses.     

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.