Studies on Tyrosinase in the Housefly

The mode of activation of protyrosinase prepared from prepupae of the housefly, Musca domestica vicina Maquart by sodium dodecyl sulfate (SDS), was studied by measuring the occurrence of tyrosinase activity over wide ranges of SDS concentrations, pH values and temperatures, either manometrically or colorimetrically. With respect to the effect of SDS concentration upon the activation of protyrosinase, it was found that there was a certain range of ratios between the concentration of SDS and that of protyrosinase which is effective for the activation. It was observed that a narrow pH range between 7 and 8 is effective for the activation. Studies of the effect of temperature on the activation indicate that the activation occurs preferentially over a limited range of temperatures. Thus, effective activation evidently occurs only with the specific experimental conditions mentioned above. These conditions suggest that a limited conformational change in the protyrosinase molecule results in the formation of tyrosinase. Precise observance of experimental conditions is required for complete activation of protyrosinase with SDS.     

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.     

Studies on Tyrosinase in the Housefly

A natural activator occurring in the aged pupae of the housefly, Musca domestica Maquart, was partially purified by means of fractionation with ammonium sulfate and of lyophilization. The preparation of the activator without accompanying tyrosinase activity made it possible to devise a method for measuring its activity, i.e., a partially purified protyrosinase which contained no activator was incubated together with natural activator, and the formation of tyrosinase activity was followed either manometrically or colorimetrically. Effects of concentration of natural activator, pH and temperature on the activation of protyrosinase were studied, resulting in an assumption that the activation occurs catalytically. Inhibition of the activation by various chemical reagents were studies and it was found that sodium picryl sulfate, N-bromosuccinimide or iodine inactivated natural activator irreversibly, and thioglycol or monoiodoacetate inhibited considerably. However, these reagents have almost no effect on the potential activity of protyrosinase. As the results, it was presumed that natural activator is an enzyme and protyrosinase is its substrate. The mode of activation of protyrosinase by the activator is, however, still obscure.     

Studies on Tyrosinase in Housefly

Occurrence of protyrosinase in the prepupae of housefly, Musca vicina Maquart, and its activation were described. The prepupae possessing no appreciable tyrosinase activity could be separated from the other aged pupae by putting them into water. Homogenate prepared from the prepupae contains protyrosinase and has no activation system for the proenzyme. As has been reported by Ohnishi²⁾ a certain activator occurred naturally in the aged pupae apparently activates the protyrosinase in vitro. However, contrary to Ohnishi’s results³⁾ it was found that this protyrosinase can be activated by the treatment with sodium dodecyl sulfate in vitro.     

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O₂-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034–13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788–6789, 2006).     

ENZYMES IN ONTOGENESIS (ORTHOPTERA) : XIII. ACTIVATION OF PROTYROSINASE AND THE OXIDATION OF ASCORBIC ACID

1. Protyrosinase from the egg of the grasshopper, Melanoplus differentialis, can be activated by excess sodium oleate or Aerosol. 2. The 3:4 quinone products of the reaction of activated protyrosinase with tyramine or tyrosine will oxidize ascorbic acid to dehydroascorbic acid. 3. The velocity of this latter oxidation of ascorbic acid increases with the amount of tyramine or tyrosine. 4. The oxidation of ascorbic acid by the tyramine-tyrosinase reaction delays the time of appearance of a red color associated with an indole quinone intermediary product in the formation of melanin. 5. Protyrosinase, in itself, and in the presence of tyrosinase substrates does not bring about the oxidation of ascorbic acid. 6. A naturally occurring substrate in a preparation of protyrosinase, sufficient to cause the oxidation of ascorbic acid, can be removed by dialysis against a 0.9 per cent sodium chloride solution. 7. Dialysis against such a solution does not change the properties of protyrosinase; the inactive enzyme must still be activated before it will catalyze the oxidation of tyramine or tyrosine. 8. When the natural substrate, tyrosine, or tyramine is absent, activation of protyrosinase does not result in the oxidation of ascorbic acid.     

Acid activation of protyrosinase from Aspergillus oryzae: homo-tetrameric protyrosinase is converted to active dimers with an essential intersubunit disulfide bond at acidic pH

Aspergillus oryzae protyrosinase (pro-TY) has a unique feature that the proenzyme is activated under conditions of acidic pH. The pro-TY was inactive at pH 7.0. The latent enzyme was activated at pH 3.0, and was slightly activated by sodium dodecyl sulfate (SDS). The molecular masses of the pro-TY and acid-activated tyrosinase (acid-TY) were 266 and 165 kDa, respectively, as estimated by gel-filtration chromatography. The CD spectra showed that the tertiary and/or quaternary structure was changed after the acid activation. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric pro-TY dissociates to dimers. Tryptophan fluorescence spectra and binding assay of 8-anilino-1-naphthalene sulfonic acid (ANS) suggested that hydrophobic amino acid residues of the active site were exposed to solvent after acid treatment. It was likely that Cys108 formed an intermolecular disulfide bond between the subunits of dimeric acid-TY. The dimerization of acid-TY involving the intermolecular disulfide bond is essential for the activity.     

ENZYMES IN ONTOGENESIS (ORTHOPTERA) : XIV. THE ACTION OF PROTEINS ON CERTAIN ACTIVATORS OF PROTYROSINASE

1. Proteins, when added to activators (sodium oleate. Aerosol) of protyrosinase, greatly decrease the degree of activation. 2. Certain proteins adsorbed on activator micelles are markedly affected by temperature and are rendered more sensitive by ultraviolet light. 3. Ideas are expressed as to the possible nature of activating and inhibiting phenomena especially as they relate to the enzyme tyrosinase.     

Isolation and characterization of the tyrosinase gene from Neurospora crassa

A precursor form of Neurospora crassa tyrosinase has been identified by Western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mRNA. The molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. In order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. Poly(A) RNA isolated from tyrosinase-induced cultures of N. crassa was used as a template for cDNA synthesis, primed by a tyrosinase-specific, 32-fold degenerate heptadecanucleotide. Based on this sequence, a unique 21-mer was synthesized and used to screen a cDNA library constructed from tyrosinase-enriched mRNA. A partial genomic DNA library from wild-type strain TS and a genomic library from strain OR were screened using a 400-base pair nick-translated SalI fragment from a tyrosinase-positive cDNA clone as hybridization probe. The DNA sequences obtained revealed the presence of two allelic forms of this enzyme. The coding regions are interrupted by two short introns, of 52 and 99 base pairs. The encoded proteins differ in 3 out of 621 amino acid residues. A comparison of the deduced amino acid sequence with the known primary structure of mature tyrosinase alleles (Rüegg, C., Ammer, D., and Lerch, K. (1982) J. Biol. Chem. 257, 6420-6426) showed that the enzyme is synthesized as a precursor. Protyrosinase exceeds the mature protein by 213 amino acids at its carboxyl terminus. The possible involvement of carboxyl-terminal processing in enzyme activation is discussed.     

Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

Laccase from a non-melanogenic,alkalotolerant gamma-proteobacterium JB isolated from industrial wastewater drained soil

A Gram-negative, alkalotolerant bacterium, isolated from the soil continually drained with industrial wastewater and identified as gamma-proteobacterium by partial 16S rRNA sequence analysis, produced a polyphenol oxidase, which showed laccase but not tyrosinase activity. The organism grew well from pH 6 to 10 and produced laccase maximally at pH 10. The enzyme was stable from pH 3 to 10.6 for at least 24 h and was optimally active at 55 °C and pH 6.5 in a 5 min assay.     

A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid.

A continuous spectrophotometric method for the rapid determination of diphenolase activity of tyrosinase is described. It uses 3,4-dihydroxymandelic acid (DOMA) as the substrate of tyrosinase and measures the final product, 3,4-dihydroxybenzaldehyde (DOBA). The spectrum of this product shows a bathochromic displacement of its absorbance maximum when the pH increases. The optimization of the method is described by using tyrosinase from several biological sources, whose enzymatic activities show different optimal pH. Thus, the enzymatic activity of mushroom tyrosinase was assayed at pH 7.5 and monitored at 350 nm (epsilon 350 pH 7.5 (DOBA) = 15,200 M-1 cm-1), whereas the spectrophotometric experiments with grape tyrosinase were carried out at pH 3.0 and monitored at 310 nm (epsilon 310 pH 3.0 (DOBA) = 9200 M-1 cm-1). The method for mushroom tyrosinase was found to be 50-fold more sensitive than the commonly used dopachrome assay, whereas for grape tyrosinase the method was found to be threefold more sensitive than the commonly used o-quinone production assay. The great solubility and stability of the chromophoric product, DOBA, as well as its high molar absorptivities at any pH, enable the method to be employed to determine the diphenolase activity of tyrosinase from different biological sources.     

Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms

A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms. Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purification. The tyrosinase in gill tissue extracts showed latent activity above pH 5.5 and suppressed or displayed no latent activity below pH 5.5 when assayed in the presence of SDS. The purified isoforms showed monophenolase activity toward 4-hydroxyanisole but practically no activity toward tyrosine or tyramine. The purified isoforms showed greater activity toward catechol than either 4-methylcatechol, dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin. The Km for catechol was similar for the group of isolated isoforms (4.3 mM) compared to the isoforms in crude extracts (5.3 mM). Crude extracts showed several isoforms ranging from 50 to 230 kDa after partially denaturing SDS PAGE, while the purified isoforms showed molecular weights of 70 kDa.     

An electrometric method for the determination of tyrosinase activity.

The pathway of dopachrome formation from L-dopa involves the net release of one proton for each molecule of dopachrome formed. The protons produced as a consequence of the enzymic step catalysed by tyrosinase can be measured by an electrometric device able to monitor changes in H+ concentration below 1 microM. This electrometric recording can be used as a simple, sensitive and continuous method for determining tyrosinase activity. The electrometric method can also be used in the presence of ascorbate by the spontaneous coupling of ascorbate oxidation to dopaquinone reduction, but measuring proton uptake instead of proton release.     

pH-dependent interconvertible forms of mushroom tyrosinase with different kinetic properties

The lag in cresolase activity and inhibition by excess tyrosine of mushroom tyrosinase which was observed when assayed at pH 6.8 was found to be absent when assayed at pH 5.0. The absence of lag and inhibition by excess tyrosine of tyrosinase at pH 5.0 were brought about only after the enzyme was kept at pH 5.0, at 0-4 degrees C, for 1.5 h. The enzyme kept at pH 5.0 for 1.5-3 h at 0-4 degrees C when brought back to pH 6.8, acquires lag and inhibition by excess tyrosine when its activity was measured at pH 6.8. The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin, and thus appear to be a general property of tyrosinase from diverse sources.     

Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH.     

Photoinactivation of agaricus bisporus tyrosinase: modification of the binuclear copper site

Irradiation of Agaricus bisporus tyrosinase in the presence of citrate at pH 5.6 with 300-420-nm light results in a loss of both catecholase activity and cresolase activity. The light-sensitive species appears to be an enzyme-citrate complex, most likely involving coordination of citrate to the active site copper. One copper ion from each binuclear active site can be removed from the inactivated enzyme, resulting in the formation of a met apo derivative. The electron spin resonance spectrum of met apo tyrosinase resembles that of met apo hemocyanin and half-met Neurospora tyrosinase. It is consistent with a distorted square-planar geometry around the copper and with either nitrogen or nitrogen and oxygen ligands. Amino acid analysis indicates that four histidines on the heavy subunit are destroyed during the inactivation process. Some or all of these histidines may serve as ligands to the copper ion which becomes labile after inactivation. Photoinactivation results in decarboxylation of citrate and does not require the presence of oxygen. The reaction may involve generation of a free radical from the citrate which then attacks nearby histidine residues.     

Melanins From Opioid Peptides

Opioid peptides and other Tyr-NH₂-terminal peptides are substrates in vitro for mushroom and sepia tyrosinase, giving rise to synthetic melanins retaining the peptide moiety (opiomelanins). The melanopeptides are characterized by a total solubility in hydrophylic solvents at neutral and basic pH. Opioid peptides (enkephalins, endorphins, and esorphins), if oxidized by tyrosinase in the presence of Dopa, are easily incorporated into Dopa-melanin, producing mixed-type pigments that can also be solubilized in hydrophylic solvents. Melanins derived from opioid peptides exhibit paramagnetism, as evidenced by an EPR spectrum identical to that of Dopa-melanin. However, the presence of the linked peptide chain is able to influence dramatically the electron transfer properties and the oxidizing behaviour of the melanopeptides, so that whereas Tyr-Gly-melanin appears to behave as Dopa-melanin, Enk-melanin does not exhibit any oxidizing activity. Opiomelanins are characterized by a peculiar UV-VIS spectrum; that is, by the presence of a distinct peak (330 nm) that disappears upon chemical treatment by acid hydrolysis. Opiomelanins are stable pigments at neutral and basic pH in the dark, whereas the addition of H₂O₂ leads to a 15% degradation. Under simulated solar illumination, opiomelanins are more easily destroyed with respect to Dopa-melanin, with increasing degradation when exposed to increased hydrogen peroxide concentrations and more alkaline pH. Some speculations on the possible existence and role of opiomelanins have been outlined.     

The melanogenic system of the liver pigmented macrophages of Rana esculenta L.--tyrosinase activity

The enzyme system responsible for Amphibian Kupffer Cell (KC) melanogenesis has not been entirely elucidated. This research demonstrates that the KC melanosomes of Rana esculenta L. possess a tyrosine-hydroxylase (TH) activity, showing that a tyrosinase is the enzyme involved in the melanogenesis. The TH reaction depends on catalytic Dopa as a cofactor and is not affected by catalase or H2O2, showing that it is catalysed by the tyrosinase and not by the peroxidase present in the melanosomes. The TH reaction is activated by Cu2+ ions but not by other tyrosinase activators such as limited proteolysis, protein ageing, and Sodium Dodecyl Sulphate (SDS). SDS inhibited the KC TH activity even below the critical micelle concentration. All these results suggest that the KC-tyrosinase differs in structure from other known tyrosinases. Using anti-KC-tyrosinase antobodies, we observed that the sites of the tyrosinase location within the cell are the same as those described in the melanocytes. In the immunoblots, the anti-KC-tyrosinase antibodies also recognised two protein bands, at the higher molecular weight ranges, in the protein electrophoretic pattern. Moreover, the tyrosinase activity was limited to the highest molecular weight band of about 260 kDa, suggesting that the enzyme activity could depend on a molecular aggregate. The melanin produced in the liver was found to be a 5,6-dihydroxyindole-rich eumelanin similar to the Sepia melanin.     

Compound Inhibitory to Clostridium botulinum Type E Produced by a Moraxella Species

A Moraxella strain, A-43, produced a compound inhibitory to the outgrowth of Clostridium botulinum type E spores. The inhibitor could be produced in various laboratory media, and the outgrowth of germinated spores was inhibited by a 1/10th dilution of the A-43 spent medium. Germination was not affected. Molecular weight of the inhibitor was estimated at 800 to 1,000. The inhibitor was dialyzable and could be concentrated by lyophilization. It was stable at 37, 25, and 5 C, but was 70% inactivated when heated at 65 C for 10 min. The inhibitor was not volatile and could not be vacuum-distilled at 40 C. Solutions of acids with pH values below 2.0 destroyed the activity. The A-43 inhibitor appears to be similar, in molecular weight and inhibition characteristics, to tylosin.