Dopa oxidase

R. Min, S. H. Lee, and Y. S. Kim. In- S1061 hibitory effect of herbal extracts of DOPA oxidase activity of tyrosinase. 

During our investigation on the metabolism of deoxyfructo-serotonin we have observed a strong inhibition of mushroom DOPA-oxidase (190 (Fig. 5). 

It has been reported that Mycobacterium leprae contains a specific DOPA-oxidase and the the metabolites of D0PA are essential for the growth of M. leprae in vivo (20). In vitro we have observed that deoxyfructoserotonin inhibits the incorporation of [ H]-D0PA in Mycobacterium leprae (21). 

Figure 5. Inhibition of DOPA-oxidase by deoxyfructoserotonin. Key 1, oxidation of DOPA by DOPA-oxidase and 2, inhibition by DFS of the oxidation of DOPA...

Mulberroside F (also known as 3 -di-0-P-D-glucopyra-noside and moracin M-6), extracted from mulberry leaves, inhibits tyrosinase activity, in the conversion of dopa (3,4-dihydroxy-P-phenylalanine) into dopachrome (dopa oxidase activity). This tyrosinase-inhibiting activity has been measured as 4.5 times more powerful than that of kojic acid. Mulberroside F also has an antioxidant activity on the superoxide anion. 

Mushroom tyrosinase was extracted as described by Ingebrigtsen and Flurkey (J. Food Sci., in press). Tyrosinase activity was monitored using either catechol, dopa or tyrosine as the substrates. All assays were carried out in the presence and absence of 0.1% SDS (w/v) to detect active and latent enzyme activities. The catechol oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 10 mM catechol and the absorbance monitored at 410 nm (25-26). The dopa oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 5 mM L-dopa and the absorbance monitored at 475 nm. The tyrosine hydroxylase activity of tyrosinase was assayed in 33 mM phosphate (pH 6.0) containing 0.33 mM L-tyrosine and the absorbance monitored at 280 nm. Protein content was determined by the method of Lowry et al. (26). 

Measurements of total tyrosinase activity do not reflect its isoenzyme composition. Therefore, we examined the isoenzyme forms of tyrosinase in small pins, large pins, immature and mature mushrooms by native electrophoresis followed by staining for dopa oxidase activity. Using dopa oxidase as an indicator of tyrosinase, one dominant isoenzyme form (I) was present in all development stages (Fig. 3). Two faster migrating forms (IIIa,b) were also apparent as well as a faint intermediate migrating form (II). No new isoenzyme forms were observed during development. 

Figure 5. Tyrosinase isoenzyme forms identified in three different breaks (1—3) after native electrophoresis and staining for dopa oxidase activity. A—D represent small pins, large pins, immature, and mature mushroom samples, respectively. (Reproduced with permission from ref. 35. Copyright 1989/. Food ScL)...

It has also been observed that tryptophan, like dopa, inhibits tyrosine hydroxylase and dopa oxidase activity of melanosomal tyrosinase and that its inhibitory mechanism differs from inhibition caused by non-substrate type compounds like cysteine and ascorbic acid (36). In fact, tyrosinase is inhibited by its own substrate in vitro and this inhibition mechanism differs from that caused by cysteine and ascorbic acid (242, 268). 

However, when Syrian (golden) hamster melanoma cells were fused with non-melanin-producing C3H mouse cells [104], and the hybrid line was isolated by the appropriate selection, no melanin production was observed in the hybrid cells, nor were tyrosinase and dopa oxidase activities detected in their extracts [104]. The parental melanoma cells had the enzymic activities and produced pigment. Nevertheless, in the hybrid cells, which possessed the expected hybrid karyotype, the lactate and malate dehydrogenase isozyme patterns were combinations of both parental types [104]. Thus, one might conclude that in the non-melanin-producing C3H parental cells there existed a specific diffusible enzyme... 

Catechol oxidases are widespread in nature. They are named according to their most important substrates as monophenol oxidases, polyphenol oxidases, phenolases, DOPA oxidases, cresolases, tyrosinases, etc. The specificity of most catechol oxidases is rather broad. 

Cornbleet melanin is produced by dendritic cells which are derived from the neural crest in the negro skin, these cells give a positive dopa oxidase reaction from the third month of fetal life. Subsequently the melanin is transferred to the cells of the Malpighian layer of the dermis. 

The papers refer mainly to the localization of the enzyme in various histological or embryological regions only a few papers made any attempt to describe the intracellular localization of the enzyme. It appears to be localized mainly in the cytoplasm and to be of two types, granular and diffuse, usually found together. There is some doubt about the dopa oxidase reaction, since no attempt seems to have been made to ensure that the reaction observed is due entirely to enzyme activity. 

Albinism, due to lack of tyrosinase and dopa oxidase, which form the natural melanin pigments of skin, hair and retina from tyrosine. The condition is unassociated with any abnormal urinary metabolite. Of these inborn errors, albinism is the most common, cystinuria is moderately rare, and the others are very uncommon. The most dangerous are porphyrinuria, with its hypersensitisation to light, and cystinuria, which tends to the formation of renal calculi. 

Winder, AJ Harris, H. New Assays for the Tyrosine Hydoxilase and Dopa Oxidase... 

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