Stimulation by ionophores of tyrosinase activity of mouse melanoma cells in culture

The hypothesis that the melanosome is an acidic vesicle in which the Tyrosinase action is suppressed under the ordinary culture conditions was examined with a variety of ionophores added in cultures of mouse melanoma cell line B16-C2M. In the presence of monensin or nigericin, which exchange H+ for Na+ or K+, respectively, through bio-membrane, the Tyrosinase activity of cells in culture was more than 10 times that in the control culture. This stimulation was observed without delay after addition of the chemicals and was not inhibited by cycloheximide. The Enzyme activity of sonicated cell-free extracts, in which melanosomes were disrupted, was not stimulated by these ionophores. The Tyrosinase activity was stimulated to a lesser extent by a proton ionophore, p-trifluoromethoxyphenylhydrazone (FCCP). The activity was also stimulated by kryptofix 221, valinomycin (Na+ and K+ carrier, respectively), and tetraethylammonium ions (permeant cations) but only in the presence of a limited concentration of FCCP. N-Ethylmaleimide and N, N'-dicyclohexylcarbodiimide, inhibitors of lysosomal Proton Pump, stimulated Tyrosinase activity of cells in the presence of FCCP. These facts are consistent with the hypothesis described above.