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Two tyrosinase isozymes were purified from pigmented hamster melanoma and injected i.v. into rats. It was shown that while T1 (sialylated) isozyme survived in the circulation, native asialo (T2) isozyme and neuraminidase-desialylated T1 isozyme disappeared from the circulation in a few minutes. Desialylated fetuin had a marked inhibitory effect on the removal of asialo-T1 tyrosinase. These data indicate that the enzyme tyrosinase shares the common pattern of clearance from circulation known for the majority of serum glycoproteins. The electrophoretic pattern of tyrosinase isozymes partially purified from the sera of melanoma-bearing animals were compared with those from the soluble fraction of the tumors. In hamsters, melanoma tissue revealed both T1 and T2 isozymes while serum exhibited T1 and very weak T2, supporting the mechanism of clearance demonstrated in rats. In mice bearing Cloudman S-91 or B-16 melanomas, only T1 isozyme was seen in sera and in tumors.

The authors point out that melanogenuria, i.e. the excretion of specific phenolic and, particulary, indole metabolites in melanoma, is undoubtedly due to elevated tyrosinase activity in this disease. Other enzymes, in particular glucuronyltransferase, catechol-O-methyltransferase and possibly hydroxyindole-O-methyltransferase, also evidently participate in the further biochemical conversion of the resultant metabolites, however.

A simple spectrophotometric method for a rapid determination of tyrosinase (EC 1.14.18.1) is described. The basis of the assay is the incubation of the enzyme with L-dopa in the presence of an optimal concentration of Zn2+ ions and the measurement of the formation of melanochrome, as indicated by the rise in absorbance at 540 nm. Final absorbance change reflects probably two activities of tyrosinase: the oxidation of dopa to dopaquinone and the conversion of 5,6-dihydroxyindole to melanochrome. Using a purified preparation from hamster melanoma, the assay was found to be more sensitive than the commonly used dopachrome assay. Comparison with some other currently available methods for assaying tyrosinase is presented and potential applications of the assay are discussed.

1. Tyrosinase was purified from melanosomal fraction of hamster melanoma. 2. A radioimmunoassay was developed to quantitate the tyrosinase protein in hamster serum and hamster melanoma tissue using polyclonal anti-tyrosinase antibodies and 125I-labeled enzyme. 3. The serum tyrosinase levels were found to be about 0.24 micrograms and 1.14-4.48 micrograms/ml in normal hamsters and melanoma-bearing hamsters, respectively. 4. Tyrosinase protein in serum correlated significantly with the enzyme activity in hamsters with melanoma (r = 0.733). 5. In the cytosol fraction of hamster melanoma, a level of 2.2 micrograms of tyrosinase/mg protein was determined. 6. The usefulness and possible applications of the tyrosinase radioimmunoassay are discussed.

The excretion of Thormählen positive melanogens (TPM), zincuria, serum dopaoxidase activity of tyrosinase and serum sialic acid were determined in 60 patients with primary and/or metastatic cutaneous melanoma and in 20 healthy persons. On the basis of our results we can recommend the following of TPM urinary excretion and serum dopaoxidase activity in the course of malignant melanoma as specific markers of the tumor growth. The following of serum sialic acid in the course of malignant melanoma is valuable from the standpoint of prognosis of the disease. The following of zincuria in the course of malignant melanoma is not recommended because of its low value for monitoring melanoma patients.

Polyclonal antibodies to hamster melanoma tyrosinase were raised in rabbits, and series of immunoinhibition experiments with a purified enzyme and specific immunoglobulins were carried out. Tyrosinase activity was determined by a set of radiochemical and spectrophotometric methods utilizing tyrosine, dopa, dopamine, or dihydroxyindole (DHI) as substrates. The quantitative data obtained indicated that the complexing of tyrosinase with its specific antibody inhibited melanogenesis in a specific manner: dopachrome formation from dopa and dopamine conversion to melanin were not affected and all other enzyme activities comprising the DHI oxidation step were inhibited to various degrees. Additionally, tyrosine hydroxylation was also slightly inhibited. The data obtained implied that melanogenesis was restricted at the point of DHI oxidation. From observations on the immunoinhibition of a DHI oxidation at varying dopa-cofactor concentrations, we propose that dopa-cofactor may be bound at separate site than DHI and thus may act as a positive allosteric effector for DHI oxidation by tyrosinase. Study of tyrosinase immunoinhibition by the antibodies against the enzyme thus seems to provide a valuable system for investigating the tyrosinase-mediated melanogenesis.