Activation of tyrosinase in mouse melanoma cell cultures.
Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37 degrees C for a minimum of 8 h. Enzyme activity continued to increase for 48 h at which time the maximal level of activation was observed. Activation did not occur at 4 degrees 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 Vmax of tyrosinase 10-fold and lowered the Km 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.
D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D007700:Kinetics; D008544:Melanocytes / Q000201:enzymology; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D014445:Tyrosine Decarboxylase / Q000378:metabolism
D004156:Catechol Oxidase; D014446:Tyrosine 3-Monooxygenase; D014442:Monophenol Monooxygenase; D014445:Tyrosine Decarboxylase