Tyrosinase Reaction Research Articles

Km of tyrosinase in microsomes in higher than that in melanosomes.

ABSTRACTKm values of tyrosinase solubilized from melanosomes and microsomes from Harding‐Passey mouse melanomas, consisting of rough and smooth surface membranes were found to be 0.6 mM and 1.3 mM, respectively. The maximum reaction velocities of the two enzymes from the melanosomes and microsomes were similar. In comparison with the tyrosine level in human plasma, reported to be approximately 0.06 mM, the Km value for microsomal tyrosinase is very high. Therefore it is likely that the tyrosinase reaction occurs at a extremely low level in the smooth and rough membranes of the melanocyte. This may be one possible reason why melanin formation does not take place in vivo in rough and smooth surface membranes in the melanocytes, despite the fact that tyrosinase activity has been detected in microsomes in vitro after isolation from melanoma tissue.

The hydroxyl radical is not involved with tyrosinase inactivation.

ABSTRACTTyrosinase [EC 1.4.18.1] in the mouse melanoma melanosome was inactivated during the dopatyrosinase reaction. When the air phase in the reaction mixture was replaced by N2 gas, tyrosinase inactivation during incubation with dopa did not occur. This may indicate that oxygen radicals and/or hydroxyl radicals (OH) are generated in the tyrosinase reaction and attack tyrosinase itself with inactivation. As shown in our previous report, oxygen radicals such as superoxide anion (O2−) and singlet oxygen (1O2) have nothing to do with tyrosinase inactivation. In this paper, we tested whether or not the hydroxyl radical could be related to the inactivation of tyrosinase. The enzyme inactivation was not prevented by D‐mannitol, a scavenger for hydroxyl radical. Furthermore, production of hydroxyl radicals which react spontaneously with methionine to yield ethylene, which can be measured by gas chromatography, was not detected in this enzyme reaction. Thus the inactivation involved is not due to the hydroxyl radical.

Inhibition of tyrosinase by indole compounds and reaction products protection by albumin

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-products(s) inhibiton or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.

Nature of Tyrosinase Inactivation in Melanosomes

The decrease in tyrosinase activity following incubation of melanosomes isolated from mouse melanoma with dopa appeared to be related to the degree of in vitro melanization of melanosomes caused by incubation with dopa. The inactivation similarly occurred in the dopa-tyrosinase reaction system containing ascorbic aicd, although in the presence of ascorbic acid the dopa-quinone is immediately converted back to dopa and therefore melanin formation does not take place. The mode of inactivation of tyrosinase in melanosomes appeared to be similar to the reaction inactivation which is known to occur in the reaction of plant plant tyrosinase. Superoxide anion (O-2) and singlet oxygen (1O2) were estimated but undetectable during the dopa-tyrosinase reaction. Thus the inactivation of tyrosinase is not caused by the reaction products or the oxygen radicals.

The dual effect of melanocyte-stimulating hormone (MSH) on the growth of cultured mouse melanoma cells

Melanotropin (melanocyte-stimulating hormone, MSH) can either stimulate or inhibit the rate of proliferation of melanocytes. Stimulation was observed in cultures of mouse melanoma cells that had low tyrosinase activity and in cells that had normal tyrosinase activity but were grown in medium that was changed frequently or was deficient in tyrosine. In contrast, inhibition of growth was associated with high tyrosinase activity and the presence of tyrosine in the culture medium. Small amounts of exogenous adenosine 3′,5′-cyclic monophosphate (cAMP) stimulate the growth rate of these cells [1]. Since MSH causes an increase in intracellular levels of cAMP 2., 3., 4., it is likely that the stimulation of growth is due to the ability of this hormone to increase the intracellular levels of cAMP. Inhibition of growth in turn is related to the activation of tyrosinase by MSH and the accumulation of toxic substances produced by the tyrosinase reaction.

The dual effect of melanocyte-stimulating hormone (MSH) on the growth of cultured mouse melanoma cells

Summary Melanotropin (melanocyte-stimulating hormone, MSH) can either stimulate or inhibit the rate of proliferation of melanocytes. Stimulation was observed in cultures of mouse melanoma cells that had low tyrosinase activity and in cells that had normal tyrosinase activity but were grown in medium that was changed frequently or was deficient in tyrosine. In contrast, inhibition of growth was associated with high tyrosinase activity and the presence of tyrosine in the culture medium. Small amounts of exogenous adenosine 3′,5′-cyclic monophosphate (cAMP) stimulate the growth rate of these cells [1] . Since MSH causes an increase in intracellular levels of cAMP 2. , 3. , 4. , it is likely that the stimulation of growth is due to the ability of this hormone to increase the intracellular levels of cAMP. Inhibition of growth in turn is related to the activation of tyrosinase by MSH and the accumulation of toxic substances produced by the tyrosinase reaction.

ELECTRON MICROSCOPIC DEMONSTRATION OF TYROSINASE IN PTERINOSOMES OF THE FROG XANTHOPHORE, AND THE ORIGIN OF PTERINOSOMES.

In the frog, Rana japonica, the successive appearance of types I, II and III pterinosomes, which were defined according to the degree of lamellar structure, is in keeping with the xanthophore differentiation at the larval stage, but these three types coexist in a single xanthophore in the adult. An intense tyrosinase reaction was found in type I-II intermediate form in the larval and adult xanthophores, but it was rarely observed in types I and III. A tyrosinase reaction was always found in the GERL (Golgi-associated Endoplasmic Reticulum) of larval and adult xanthophores, and it was similarly evident in small Golgi vesicles which were separated from the GERL and dispersed in the cytoplasm. The above findings suggest that tyrosinase and pterinosome originate from different parts of the cytoplasm. The hypothesis that small Golgi vesicles are transported to the tyrosinase-negative premelanosomes involved in the origin of the melanosome is also applicable to the origin of pterinosomes.

Metabolism of tyrosine by Microspira tyrosinatica: a new intermediate [2,4,5-trihydroxyphenylalanine (6-hydroxydopa)] in the tyrosinase reaction.

Metabolism of tyrosine by Microspira tyrosinatica: a new intermediate [2,4,5-trihydroxyphenylalanine (6-hydroxydopa)] in the tyrosinase reaction.

Effect of catechol-thiol conjugates on tyrosinase-dependent tyrosine hydroxylation.

1. The tyrosinase reaction in the presence of a thiol compound was studied using mushroom tyrosinase (EC 1.10.3.1) with regard to catechol-thiol conjugates. 2. Although tyrosine hydroxylation of tyrosinase was extremely decreased in the presence of a thiol compound, the inhibitory effect was removed by the addition of a pyrocatechol-cysteine conjugate, S-(2,3-dihydroxyphenyl) cysteine, which was not oxidized by the enzyme. 3. The pyrocatechol-cysteine conjugate was also able to shorten the lag period of tyrosinase-dependent tyrosine hydroxylation. 4. The sigmoidal reaction curve of tyrosine hydroxylation observed in the presence of sulfhydryl compounds was found to be caused by the catechol-thiol conjugates, the final products of the enzyme reaction, which counteract the inhibitory effect of sulfhydryl compounds. 5. The pyrocatechol-cysteine conjugate, on the other hand, was shown to cause the decrease of the reaction rate of the enzyme during incubation.

Inactivation mechanism of tyrosinase.

Soluble tyrosinase isolated from the Harding-Passey mouse melanoma was incubated with 14C-dopa and the time course of disappearance of dopa was measured by radioactivity assay at various tyrosinase concentrations. At the end of certain incubation periods, the tyrosinase activity was lost and the dopa concentration reached at stationary levels depending on the initial tyrosinase concentrations. The kinetic studies were carried out in order to analyze the relationship between the amount of tyrosinase incubated and the amount of dopa disappeared at various incubation periods. The results of the kinetic treatments indicated that inactivation of tyrosinase occurred mainly in proportion to the total concentration of tyrosinase present and the inactivation due to the binding with reaction products did not seem to play an important role in the mechanism. In other words, the inactiva-tion mechanism involved appears to be simlar to the reaction inactivation, which is known to occur in the reaction of plant tyrosinase.

Suppression of pigmentation in mouse melanoma cells by 5-bromodeoxyuridine: effects on tyrosinase activity and melanosome formation.

Low concentrations (1-3 microg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B(5)59) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C(3)471), derived by long-term culture of B(5)59 cells in 1 microg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B(5)59 cells during a 7-day period of growth with BrdU (3 microg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T(1) and T(2) forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T(1)) was consistently affected earlier than the other (T(2)). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process.

Protein transport in mouse kidney utilizing tyrosinase as an ultrastructural tracer.

The morphological basis of glomerular filtration and protein reabsorption in mouse kidney was examined by using mushroom tyrosinase subunits (mol wt 34,500), as an ultrastructural tracer. Almost immediately after injection tyrosinase reaction product was visualized in the glomerulus, and within the capillary lumen extending into the endothelial fenestrae. The entire basement membrane showed accumulations of tyrosinase in the subendothelial and subepithelial layers. The urinary space contained considerable amounts of reaction product, some of which was adsorbed to the cell coat of the podocytes. Reaction product could also be seen in the brush border region of the proximal tubule cells. By 30 min after injection, no tyrosinase reaction product was demonstrable in the glomerulus except for dense vesicles in mesangial cells. Most of the reaction product was localized in absorption droplets in the apical cytoplasm of proximal tubule cells. Occasionally, some tyrosinase reaction product was present within the basal infoldings of these cells. The behavior of tyrosinase in the mouse kidney is in accordance with that of other low molecular weight tracers. The pattern of localization within the basement membrane provides additional support for the presence of two filtration barriers in the glomerulus. The adherence of tyrosinase to the cell coat of the glomerular epithelial cells suggests that this may be an additional mechanism whereby protein is removed from the glomerular filtrate. Tyrosinase subunits may prove to be a useful new tracer for the study of protein transport.

Fate of l-[3,5- 3H]tyrosine in cell-free extracts and tissue cultures of melanoma cells: A new assay method for tyrosinase in living cells

On incubation of l-[3,5- 3H]tyrosine (1 m m) with a cell-free extract of cultured melanoma cells (cell line C 2M) in the presence of dopa (0.1 m m), tritium was released as water at a steady rate for about 15 hr, or until 20% of the radioactivity had been converted to water. Some radioactivity was also found in dopa and melanin, but no appreciable amount in any other compounds. A cell-free extract of amelanotic cultured cells (cell line C 2W) did not produce tritiated water or melanin. When C 2M cells were cultured in Eagle's minimum essential medium containing l-[3,5- 3H]tyrosine, they produced tritiated water, melanin and protein, but no substantial amount of dopa, while C 2W cells produced only tritiated protein. The tyrosinase reaction proceeded linearly for 24 hr in growing cultures. During incubation of a cell-free extract of C 2M cells or cultivation of these cells, the ratio of the sum of the radioactivities of water, dopa and melanin i.e., the radioactivity disappearing from tyrosine for melanin synthesis, to that of water, Δ Ty W , was fairly constant, being 1.4 for the cell-free extract and 1.1 for the culture. The values of the ratio, Δ Ty W , were very similar for reactions with a wide range of reaction rates and with different sources of cell-free extract, including human melanoma. Using the ratio Δ Ty W , the tyrosinase activity in intact cells in culture as well as in cell-free extracts can be determined as the rate of hydroxylation of tyrosine simply by assaying the radioactivity of the water produced.

Biochemical Basis for Depigmentation of Skin by Phenolic Germicides

Occupational exposure to germicides containing various phenols produces depigmentation of human skin. The chemical basis for this depigmentation may be competitive inhibition of the enzyme tyrosinase. In the tyrosinase reaction tyrosine has K m of 2.2 × 10 -3 M: p-tert -butylphenol has a K 1 of 1.95 × 10 -4 M. In the oxidase reaction, the estimated K 1 for p-tert -butylphenol is 2.02 × 10 -4 M. It is recommended that chemicals to be included in products that will come into close contact with human skin should be tested for their ability to inhibit tyrosinase.

Physicochemical and Kinetic Properties of Mushroom Tyrosinase

Abstract Tyrosinase (o-diphenol:O2 oxidoreductase, EC 1.10.3.1) has been purified from a commercial lyophilized powder prepared from edible mushrooms. Specific activities toward dl-dihydroxyphenylalanine and catechol, homogeneity, molecular weight, absorption spectrum, amino acid composition, and copper content of the preparation are comparable to other purified samples of tyrosinase prepared from fresh mushrooms. Circular dichroism of the enzyme reveals an unusually shaped envelope of negative ellipticity bands in the deep ultraviolet (196 to 250 mµ), which is difficult to explain by a combination of the usual peptide conformational forms. Small positive ellipticity bands ([θ] per 32,000 mol wt ≅ 30,000) are associated with the near ultraviolet absorption of the aromatic amino acid side chains. Kinetic constants for the oxidation of a homologous series of catechol substrates substituted in position 4 by groups —H, —SCN, —COCH3, —CHO, —CN, and —NO2 have been determined. As the electron-withdrawing ability of the substituent increases, Km and kcat decrease in the order H g SCN g COCH3 g CHO g CN g NO2. Except for the kcat values for the two most slowly oxidized substrates, both Km and kcat conform to Hammett relationships when the substituent parameter σ- is used. The ρ values are -1.01 ± 0.09 for Km and -2.49 ± 0.11 for kcat. The oxidation of pyrocatechol, 4-COCH3, and 4-CHO-catechol give a satisfactory isokinetic relationship between ΔH‡ and ΔS‡. These findings suggest that these substrates are oxidized by the same basic mechanism. Studies of the dependence of catechol oxidation on the concentration of oxygen show that Km for oxygen varies with the nature of the catechol substrate. With the exception of the pyrocatechol complex, the enzyme-substrate complexes for this series of catechols are saturated in air (20.95% O2). The oxygen kinetics suggest a sequential mechanism for the binding of catechol and oxygen. Detailed studies of the pH dependence of Km and kcat show the presence of a group in the enzyme whose pKa changes upon the binding of a catechol molecule. Inhibition of tyrosinase by benzoic acid and cyanide show that the former is competitive with catechol and noncompetitive with oxygen, while the latter is competitive with oxygen and noncompetitive with catechol. These results are taken to indicate two distinct substrate binding sites on the enzyme, one with a high affinity for aromatic compounds including phenolic substrates, the other for metal-binding agents and oxygen. The latter site presumably involves enzyme copper. Binding studies with 14C-benzoic acid suggest that there are two binding sites for benzoic acid per mole of tetramer (mol wt = 128,000). The equilibrium constant for this binding is the same as the Ki measured for competitive inhibition of catechol oxidation by benzoic acid. Catechols are activators of the phenolase reaction of tyrosinase, and tyrosine is shown to be an inhibitor of this activation of its own oxidation in a manner suggesting that there is a third binding site on the enzyme for an activator catechol or phenol.

Immunoelectrophoresis of proteins in the hemolymph of the large milkweed bug, Oncopeltus fasciatus (Dallas)

1. 1. Using immunoelectrophoresis at least eleven antigens were detected in hemolymph from fourth-instar nymphs, from male and female fifth-instar nymphs 1–8 days post-molt, from male and female adults 1–14 days post-molt, and in the egg. A minimun of five and as many as ten antigens were observed in preparation of first-, second- or third-instar nymphs. Fusion was observed between corresponding precipitin arcs containing antigens from insects of both sexes and all physiological ages studied as well as from the egg. 2. 2. Five of the precipitin arcs gave an esterase reaction which was inhibited by 10 −4 M eserine. 3. 3. Two of the precipitin arcs which stained for esterase also gave a tyrosinase reaction. 4. 4. One of the tyrosine and esterase positive arcs bound-dye specific for lipoprotein and also reacted as a glycoprotein. 5. 5. Some of the protein present bound evans blue, but it did not have the mobility characteristic of human serum albumin. 6. 6. Although the protein concentrations do change during development, the data support the idea that the proteins of the milkweed bugs in different developmental stages do not differ qualitatively in a significant manner.

Embryonic Development of Melanocytes in Human Hair and Epidermis: Their Cellular Differentiation and Melanogenic Activity

The biological and enzymic behaViDr of embryonic melanocytes and their cellular interaction in differentiating follicular and epidermal structures after leaving their dermal environment through the penetration of the dermalepidermal junction has not been significantly studied, although their neural crest origin (1) and developmental process within the dermis including their epidermal entry (2) is well described. Moreover, the melanocyte systems of adult human hair follicles excluding the infundibulum have been found to be different from that of the epidermis in their melanin synthesizing activity. The melanocyte in the peripheral layer of the outer root sheath below the melanogenic level has been described as being an amelanotic melanocyte (3) based on its negative reaction to dopa and Masson's premelanin technic. Furthermore, the hair bulb melanocyte has been shown to differ from the epidermal melanocyte in being not only dopa but also tyrosinase reaction positive in the anagen stage of the follicle (4). The origin and developmental genesis of these distinctive hair and epidermal melanocyte systems, however, have not been studied in relation to embryonic hair and epidermal differentiation despite its biological importance. Previous embryonic studies were mostly morphological characterizations which did not present information on the melanogenic activity of these developing melanocytes. In the present study the cellular differentiation, tyrosinase synthesis, and the formation of premelanin positive melanosomes and their melanization have been investigated in the melanocytes of developing embryonic human hair follicles and epidermis.

Cholinesterase in Junction Nevus

!The neural origin of all intradermal nevus cells has been proposed on the basis of their cholinesterase activity and lack of tyrosinase in contrast to the junction nevus cell (1, 2). However, tyrosinase studies of intradermal nevi after ultraviolet irradiation reveal not only an intensified dopa reaction but also a positive tyrosinase reaction in large epithelioid A-type and small B-type intradermal nevus cells, in addition to their cholinesterase activity (3). Furthermore, electron microscopy of intradermal nevi demonstrates the presence of an enzymically active melanosome system in the cytoplasm of these cells (4).

Competitive inhibition of mammalian tyrosinase by phenylalanine and its relationship to hair pigmentation in phenylketonuria.

IN Jervis's introductory study, and in subsequent case reports of phenylketonuria (phenylpyruvic oligophrenia), a blonde hair colour and a fair skin have been noted in the large majority of the patients1–4. The most striking example described by Jervis was “an idiot baby with blonde hair and blue eyes who belonged to a family of Sicilian extraction, all the members of which, for at least three generations, were of a very dark Mediterranean race”1. Three possible hypotheses may be advanced to explain the reduction of melanin formation: absence of the melanin-forming enzyme, tyrosinase; absence or decrease of the melanin precursor, tyrosine; and finally, inhibition of the tyrosine–tyrosinase reaction by phenylalanine or related abnormal aromatic metabolites which accumulate in the blood of patients with phenylketonuria.

Pathogenicity of tyrosinase-deficient mutants of Streptomyces scabies.

Natural and X-ray induced mutations rendered strains of Streptomyces scabies simultaneously tyrosinase-deficient and unable to form a "brown-ring" in skim milk. These results and the presence of the tyrosinase reaction in each of 80 brown-ring positive streptomycetes tested were considered to indicate the identity of the brown-ring test and the tyrosinase reaction. All 12 tyrosinase-deficient mutants tested were virulent for Katahdin potatoes in controlled greenhouse tests.