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Validity of the tritiated water assay technique for tyrosine hydroxylase activity as a qualitative method was demonstrated with mushroom tyrosinase. Using this method, isolated murine melanoma "tyrosinase" (L-dopa oxidase) showed no tyrosine hydroxylase activity. This finding supports previous studies in our laboratory which used a variety of histochemical and biochemical methods. The nonenzymatic production of tritiated water caused by tritium exchange with hydrogen peroxide complicates the use of the tritiated water assay technique with crude systems, since hydrogen peroxide is generated by a variety of oxidase reactions. For this reason, previous studies using the tritiated water assay technique with crude systems are ambiguous.

Melanosomal "tyrosinase" (L-dopa) was isolated from trypsin digest of B-16 mouse melanoma melanosomes, using polyacrylamide gel disc electrophoresis. The enzyme was represented by a single band, having characteristics similar to the T1 dopa-positive band observed when using supernatants of crude melanoma homogenates as the source. When gels with this band were incubated in solutions containing tyrosine and dopa in varying ratios , there was no enhancement of melanin formation by tyrosine when compared with incubations in corresponding concentrations of dopa alone. These data further support previous studies in our laboratory demonstrating an inability of so-called mamalian "tyrosinase" to convert tyrosine to melanin; since this enzyme readily converts L-dopa to melanin, it seems more reasonable to term this enzyme an L-dopa oxidase.