Activation of tyrosinase in mouse melanoma cell cultures

Summary Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37°C for a minimum of 8 h. Enzyme activity continued to increase for 48h at which time the maximal level of activation was observed. Activation did not occur at 4°C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation process increased the V _Max of tyrosinase 10-fold and lowered the K _M by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole. The “self-activation” response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells. This investigation was supported by Public Health Service grants CA41425 and CA30393 awarded by the National Cancer Institute, Bethesda, MD and by a research grant from the Proctor and Gamble Company.