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

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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.