Mammalian tyrosinase; the relationship of copper to enzymatic activity.

Copper has been reported to be an essential part of the enzyme tyrosinase prepared from various plant and insect sources (l-8). In the case of potato tyrosinase, Kubowitz (1, 2) found that the enzyme could be inhibited by reagents which combine with copper, e.g. diethyldithiocarbamate, salicylaldoxime, and carbon monoxide. He demonstrated that treatment of the enzyme with cyanide followed by dialysis resulted in a decrease in the copper content of the enzyme preparation and a loss of enzymatic activity. Addition of sufficient cupric ions resulted in practically complete restoration of activity. Other metals (iron, cobalt, nickel, manganese, and zinc) were ineffective in restoring enzymatic activity. Allen and Bodine (3) carried out experiments on protyrosinase from grasshopper eggs similar to those of Kubowitz and obtained essentially the same results. Several investigators have shown that copper is associated with the activity of phenoloxidases from mushrooms and tea leaf (4-8). Hogeboom and Adams (9) demonstrated an inhibition by cyanide of the enzymatic oxidation of L-tyrosine and dihydroxyphenyl-L-alanine (dopa), using extracts from the Harding-Passey mouse melanoma; they concluded that the enzymes concerned in the oxidation of tyrosine and .dopa by melanotic tissue are ironor copper-containing enzymes. Cunningham (10) and Greenstein et al. (11) found that copper is present in human melanomas. The experiments reported here present further evidence that mammalian tyrosinase prepared from the Harding-Passey mouse melanoma is an enzyme which requires copper for activity. This evidence is based on the demonstration that copper can be removed from the enzyme preparation with cyanide to produce a relatively inactive enzyme and that the addition of copper to such a preparation restores activity. Furthermore, sub-