Presence Of Dopa Research Articles

Effect of DOPA-Loading on Glutathione-dependent 5-S-Cysteinyldopa Genesis in Melanoma Cells in Vitro

The effect of dopa, cysteine, and glutathione on 5-S-cysteinyldopa (5-S-CD) genesis in melanoma cells cultured in normal and tyrosine- and cysteine-free media has been studied. In normal media only melanotic melanoma cells have been found to secrete 5-S-CD into the medium. In the presence of dopa and cysteine, both, media incubated with and without cells have been found to produce 5-S-CD. In the presence of dopa and glutathione, however, cell-free media did not show the presence of 5-S-CD. In contrast melanoma cell-cultured media has been found to contain large quantities of this amino acid. The optimum condition for maximum production of 5-S-CD via glutathione-dependent pathway has been found to be at the dopa concentration of 10(-5) M when glutathione is present at the concentration of 10(-5) M in the culture medium. Thus dopa concentration with regards to glutathione is 1:1 on the molar basis which is twice the dopa concentration required in in vitro noncellular tyrosinase system. It is suggested that higher dopa requirement in our melanoma cell culture system reflects the co-existence of eu- and pheomelanin synthesis taking place according to their genetically predetermined proportions.

5-S-cysteinyldopa as a substrate for tyrosinase

Oxidation of 5-S-cysteinyldopa by mushroom tyrosinase was studied by measuring 5-S-cysteinyldopa consumption with HPLC. 5-S-cysteinyldopa was found to be a substrate for tyrosinase, but our results suggests that a self-catalysed oxidation induced by enzymatically formed 5-S-cysteinyl dopaquinone also takes place. Dopa oxidation by tyrosinase was determined by measuring substrate consumption with HPLC. The oxidation of 5-S-cysteinyldopa was markedly accelerated in the presence of dopa. This could be explained by dopaquinone oxidation of 5-S-cysteinyldopa, which has a lower oxidation potential than dopa.

Periostracin ? A soluble precursor of sclerotized periostracum inMytilus edulis L.

Periostracin is a soluble presclerotin of the periostracum ofMytilus edulis. Periostracin is unusually unstable in vitro. It was extracted with high yield from marginal periostracum with pure formic acid. It is a basic, water-insoluble protein with a molecular weight of 20,000 as determined by gel electrophoresis in sodium dodecyl sulfate. The amino acid composition of periostracin is unique with 55% of the residues being glycine, 10% tyrosine and 2.2% Dopa. The presence of Dopa was further substantiated by reaction with ethylenediamine, by difference spectra at various pH and by UV and visible spectra of the oxidation products. The origin of Dopa in periostracin seems linked to a tyrosine hydroxylase active toward protein tyrosyl residues.

Molecular and ultrastructural characterization of the egg capsule of the leech Erpobdella octoculata L.

1. 1. The high content of proline and of bulky amino acid residues and the amorphous X-ray diffraction pattern suggest that much of the protein of the egg capsule of Erpobdella octoculata is amorphous. 2. 2. The sharp thermal transition, the presence of banded fibrils apparently composed of 15 Å filaments and the amino acid composition of the trichloracetic acid extract may indicate that a small part of the protein is in the form of fibrous molecules, possibly of an α fibrous protein. 3. 3. The insolubility of the material and the presence of DOPA and orthodiphenol oxidase activity suggest stabilization by phenolic cross-linking.

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.

メラニン生成機構にかんする2，3の問題

The recent availability of cell biochemical techniques as ultracentrifugation separation of cell particles and density gradient centrifugation made possible the microdissection of and biochemical studies on melanin-forming cells which have elucidated the steps involved in the formation of melanosomes. A series of experiments along the studies on melanogenesis has been carried out within the last several years. Experimental data obtained from these studies are all compartible with the hypothesis, melanosome concept that tyrosinase is synthesized in ribosomes, transferred via endoplasmic reticulum to the Golgi area, where tyrosinase is separated into small units, each of which is surrounded by a membranous envelope. Within each envelope, the tyrosinase molecules assume an ordered pattern, after which melanin biosynthesis begins and the particle is known as a melanosome. As melanin gradually accumulates, the melanosome is transformed into a uniformly dense and structureless particle, a mature melanosome. The problems described herein are the in vitro experiments on the melanization process of melanosomes and the controling mechanism of thiol-group in melanin formation in melanocyte. The reciprocal relationship between tyrosinase activity and melanization of melanosomes has been demonstrated and data presented herein would be additional evidence to support this concept. Two kinds of melanosomes in defferent developmental stages were prepared from the mouse melanoma. Melanosome No. 1 is less melanized and possesses more tyrosinase activity than melanosome No. 2. Comparison of the rate of incorporation of 14C-DOPA in vitro by melanosome No. 1 and No. 2 was carried out. The experimental results showed that melanosome No. 1, immature melanosome can synthesize and deposit more melanin than melanosome No. 2, mature melanosomes. The melanization mechanism of melanosomes was further studied. Melanosomes isolated from the mouse melanoma were incubated with the excess amount of 14C-DOPA. The amount of melanin formed was dependent on the amount of melanosomes incubated but relationship between them was an upward convex rather than a straight line. It was assumed that there are some kinds of mechanisms which inactivate the enzyme. Kinetic studies were carried out in order to analyze the kind of inactivation mechanisms thought to be present. The exprimental results obtained could not be explained by assuming only one type of inactive enzymeproduct complex. However, they could be explained on the basis of simultaneous formation of two or more types of inactive enzymn-product complex. The sulfhydryl group has been considered as one of the important controling factors in melanin formation in melanocytes. The inhibitory effects of thiol compounds ont yrosine hydroxylase and tyrosinase in melanosomes isolated from mouse melanoma are descrided and an in vitro study of the reaction between an oxidation product of DOPA and glutathione in the presence of mammalian tyrosinase is reported. The formation of new chemical compounds resulted from the presence of DOPA, glutathione and mammalian tyrosinase in solution at pH 6.8. The existence of chemical bonding between glutathione and an oxidation product of DOPA, probably DOPA-quinone was demonstrated. The glutathione-derived reaction product remained soluble in the reaction medium and did not precipitate on the melanosomes. The pKi values of glutathione and cysteine on tyrosine hydroxylase were much smaller than those on tyrosinase. The inhibition of tyrosine melanin formation by sulfhydryl compounds may take place in two ways: by inhibition of the enzyme tyrosinase, and by the formation of new compounds in which intermediates of the tyrosine-tyrosinase reaction are trapped by the sulfhydryl compounds. (to be cotinued)

Carotid body catecholamines. Histochemical studies on the effects of drug treatments.

Histochemical studies were made in the cat's carotid body using two fluorescence techniques: the formaldehyde condensation (HCOH) method and the trihydroxyindole (THI) method. A number of pharmacologic agents known to alter catecholamine metabolism or binding were used to evaluate their effect on the amines found in the glomus cells. After treatment with reserpine, both histochemical techniques showed a reduction and eventual disappearance of fluorescence from the glomus cells. Treatment with a monoamine oxidase inhibitor (iproniazid) or with dopamine beta hydroxylase inhibitor (disulfiran) enhanced the glomus cell fluorescence. The observed increase was greater with the THI than with the HCOH technique. A few yellow fluorescent cells were found following a combination of reserpine and iproniazid treatments. A reduction in fluorescence with both techniques was obtained following DOPA decarboxylase inhibitors (MK-485). It is concluded that some of the glomus cells contain only dopamine while others contain norepinephrine or a combination of norepinephrine and dopamine. In addition the presence of DOPA in some cells following treatment with pharmacologic agents may account for some of the results. Finally, the few yellow fluorescent cells found probably contain 5-hydroxytryptamine.

Catecholamine binding complex in bovine adrenal medulla: Possible involvement of calcium

Seventy per cent of the calcium of chromaffin granules of bovine adrenal medulla was estimated to be nonexchangeable by incubation of chromaffin granules with 45Ca for 19 hr at 4°. By contrast, only 34 per cent of the calcium of a preparation from bovine adrenal medulla containing lysosomes and mitochondria, as well as chromaffin granules, was nonexchangeable. These results indicate that a relatively large amount of calcium is held in a nondiffusible form within chromaffin granules and may be part of the binding complex for catecholamines in adrenal medulla. Stimulation of bovine adrenals with acetylcholine in the presence of DOPA and 45Ca resulted in a greater accumulation of 45Ca in granule fractions than stimulation with acetylcholine in the absence of DOPA. This finding suggests that calcium is incorporated into chromaffin granules during catecholamine synthesis and supports the hypothesis that calcium is part of the catecholamine storage complex in adrenal medulla.

The Role of Peroxidase Vs. The Role of Tyrosinase in Enzymatic Conversion of Tyrosine to Melanin in Melanocytes, Mast Cells and Eosinophils

Autoradiographic-histochemical observations, correlated with previous histochemical and biochemical studies in our laboratory, and with studies with isolated mammalian peroxidase, have provided evidence that mammalian peroxidase can mediate the enzymatic conversion of tyrosine to melanin, in the presence of dopa as co-factor. Our studies provided no conclusive evidence that mammalian tyrosinase has significant ability to oxidize tyrosine to melanin, although they showed that this enzyme has strong dopa oxidase activity. It is suggested that peroxidase may be the enzyme initiating melanin synthesis in vivo and that tyrosinase may utilize dopa synthesized by enzymatic activity of peroxidase. Tyrosinase may therefore function primarily as a dopa oxidase, as conceived by Bloch.

Influence of Cecropia oil on cuticular phenols in Tenebrio molitor

The juvenilized cuticle on adult Tenebrio molitor produced by intradermal injection of Cecropia oil into 1-day-old pupae was utilized for studying the influence of hormonal imbalance on the cuticular phenols. Histochemical methods revealed the absence of o-diphenols from juvenilized patches, but proved their presence in larval and pupal epicuticle and adult exocuticle. Juvenilized as well as adult cuticle were lacking in tyrosine, whereas larval and pupal exocuticle contained this compound in protein-bound form. Hence, application of a juvenile hormone-active preparation upsets the normal tyrosine transformation in the integument. It was shown by a colorimetric method that juvenilized cuticle contains as little as 1·6 μg diphenols/mg of cuticle, partially sclerotized cuticle contains 3·5 μg diphenols/mg, whereas completely sclerotized cuticle comprises 8·8 μg of diphenols/mg cuticle. The low diphenol level of the pupal cuticle appeared to be restricted to its external layers. Spectral indication for the occurrence of protocatechuic ( o-dihydroxybenzoic) acid in sclerotized cuticle of adults and the presence of dopa or N-acetyldopamine in unsclerotized adult cuticle was obtained. Thin-layer chromatography of an aqueous extract from sclerotized adult cuticle gave a single spot whose R f coincided with those of protocatechuic or 3,4-dihydroxyphenylacetic acid. A considerable amount of diphenols was demonstrated in the fully sclerotized cuticle of aged beetles. The possibility of dihydroxyphenolic acids being breakdown products of dopa was discussed.

Fluorescent Dopa Reaction of Nevi and Melanomas

In specimens treated according to the histochemical method of Falck and Hillarp, a specific fluorescence develops in melanocytes, nevus cells, and cells from malignant melanomas. There is support for the presumption that the fluorescence indicates the presence of dopa. The fluorescence of the epidermal melanocytes is faint. The fluorescence of dermal nevus cell aggregates is moderate in the parts adjacent to epithelium, whereas inner and deep parts fluoresced only faintly or not at all. The fluorescence of junctional nevus cells is more intense. Most of the cells in primary malignant melanomas and cutaneous metastases show a strong fluorescence, and single invading melanoma cells can easily be distinguished. The fluorescence intensity of melanoma cells of lymph node metastases varies within wide limits.

Fluorescent dopa reaction of nevi and melanomas

In specimens treated according to the histochemical method of Falck and Hillarp, a specific fluorescence develops in melanocytes, nevus cells, and cells from malignant melanomas. There is support for the presumption that the fluorescence indicates the presence of dopa. The fluorescence of the epidermal melanocytes is faint. The fluorescence of dermal nevus cell aggregates is moderate in the parts adjacent to epithelium, whereas inner and deep parts fluoresced only faintly or not at all. The fluorescence of junctional nevus cells is more intense. Most of the cells in primary malignant melanomas and cutaneous metastases show a strong fluorescence, and single invading melanoma cells can easily be distinguished. The fluorescence intensity of melanoma cells of lymph node metastases varies within wide limits.

MAMMALIAN TYROSINASE: MELANIN FORMATION BY ULTRAVIOLET IRRADIATION

Raper 1 and his associates, in a comprehensive study of the tyrosine-tyrosinase reaction, showed that the enzyme tyrosinase obtained from plant sources catalyzes the conversion of tyrosine to dopa (3,4-dihydroxyphenyl-l-alanine). Dopa is then oxidized, in the presence of the same enzyme, to a red indole derivative, which changes spontaneously to melanin. Melanin formation in mammals has remained somewhat of an enigma, despite the demonstration by Bloch 2 of an intracellular oxidase which catalyzes the oxidation of dopa to melanin but is without apparent effect on tyrosine. The presence of dopa in mammalian tissue or fluids has never been demonstrated, and it has not been known how tyrosine is converted into dopa. Recently, however, the enzymatic formation of melanin from tyrosine has been conclusively demonstrated in several types of mammalian tissue, including beef ciliary bodies 3 and human, 4 horse 5 and mouse 6 melanomas. The enzyme has been shown to