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Dopachrome conversion factor is an enzymatic activity associated with the pigmentary system which catalyzes the conversion of dopachrome, an intermediate in melanin biosynthesis, to dihydroxyindole-2-carboxylic acid (DHICA). To date, the mechanism of action of DCF has been unknown because all previous assays have employed a dopachrome substrate contaminated with L-dopa. It has therefore not been possible to determine whether L-dopa acts as a hydrogen donor in the reaction or whether the formation of DHICA occurs through an isomerization of dopachrome. In this study it is shown that DCF catalyzes the conversion of dopachrome to DHICA equally well in the presence or absence of L-dopa. The DCF-mediated reaction thus appears to be an isomeric rearrangement of hydrogen ions from one portion of the dopachrome molecule to another. The results indicate that the name "dopachrome isomerase" appropriately describes the function of DCF.

The biosynthesis of melanin is initiated by the catalytic oxidation of tyrosine to dopa by tyrosinase in a reaction that requires dopa as a cofactor. Tyrosine then catalyzes the dehydrogenation of dopa to dopaquinone. The subsequent reactions can proceed spontaneously in vitro. Tyrosinase, purified from murine melanomas and the skins of brown mice, has now been shown to catalyze a third reaction in mammalian melanogenesis, namely the conversion of 5,6-dihydroxyindile to melanochrome. This reaction requires dopa as a cofactor and is inhibited by tyrosine. Conversely, 5,6-dihydroxyindole inhibits the oxidation of tyrosine to dopa, so that the relative concentrations of tyrosine and 5,6-dihydroxyindole within the mammalian pigment cell are capable of regulating melanogenesis in a previously unrecognized fashion. Tyrosinase has the unusual property of catalyzing three distinct reactions within a single biochemical pathway: the hydroxylation of a monophenol, the dehydrogenation of a catechol, and the dehydrogenation of a dihydroxyindole. The first and third of these reactions require dopa as a cofactor; in the second reaction, dopa is a substrate.

Three new regulatory factors in the melanogenesis pathway were recently described: dopachrome conversion factor accelerates the conversion of dopachrome to 5,6-dihydroxyindole; indole conversion factor accelerates the conversion of 5, 6-dihydroxyindole into melanin; and indole blocking factor retards the conversion of 5, 6-dihydroxyindole into melanin. Exposure of wild-type Cloudman melanoma cells in culture to melanotropin (MSH) removes blocking factor activity and increases indole conversion factor activity. The chemical nature of factors has not yet been determined. In this report we demonstrate that highly purified isozymes of tyrosinase from C57B1/6N murine hair bulbs and B16 murine melanoma are closely associated with conversion and blocking factor activities. The soluble isozymes. T1, T2, and T2 contain blocking factor activity, while isozyme T4, the major tyrosinase species found in melanosomes, contains activity that accelerates melanin formation from dopachrome. The results suggest that melanogenesis is regulated by the association of these different factors with the specific tyrosinase isozymes.

Modern techniques offer an opportunity for a more complete evaluation of melanin production in the uvea and retinal pigment epithelium (RPE). By measuring the release of tritium from tritiated tyrosine in homogenized samples of adult bovine RPE as well as iris and choroid, tyrosinase activity could be demonstrated in both the uveal tract and the RPE. Phenylthiourea, a specific tyrosinase inhibitor, markedly decreased tyrosinase activity, whereas 3-iodo-tyrosine, a tyrosine hydroxylase inhibitor, had no effect. These techniques indicate tyrosinase activity in the uveal tract and the RPE of adult cattle. This is the first biochemical demonstration of tyrosinase in adult RPE.

Melanocyte stimulating hormone coupled to Sepharose effects an increase in tryosinase (EC 1.14.18.1; monophenol monoxygenase) activity of cultivated mouse melanoma cells. Synchronized cells are found to respond to melanocyte stimulating hormone only in the G2 phase of the cell cycle, although their response to cyclic AMP is independent of position in the cell cycle. The binding of (125)I-labeled melanocyte stimulating hormone occurs predominantly in G2. These observations are satisfied by a model in which the hormone can activate adenylate cyclase (EC 4.6.1.1) by binding to a melanocyte stimulating hormone receptor only in G2; the events distal to cyclic AMP production can occur throughout the cell cycle.

L-tyrosine, a precurosr to melanin, has recently been shown to be a regulator of the melanogenic pathway in some cultured melanoma cell lines. In this paper we demonstrated that L-tyrosine, besides increasing binding capacity for MSH, decreased cooperativity between MSH receptors and increased the level of tyrosinase induction by MSH. Apparently, regulation of MSH receptor activity by L-tyrosine involves specific changes in the interactions between the receptors and modification of the cellular responsiveness to MSH.