Functional analysis of tyrosinase isozymes of cultured malignant melanoma cells during the recovery period following interrupted melanogenesis induced by glycosylation inhibitors.

Multiple forms of tyrosinase, T1, T2, T3, have been shown to differ with respect to carbohydrate moieties of these isozymes. We demonstrated that, in cultured B-16 melanoma cells, melanization can be completely interrupted by glycosylation inhibitors, such as glucosamine and tunicamycin, and that these inhibitors cause a selective loss of membrane-bound T3. It is further found that inhibition of melanization induced by glucosamine occurs even in the presence of protease inhibitors, such as phenylmethylsulfonyl fluoride and leupeptin, and that melanization inhibition is reversible upon removal of the inhibitor. In this report we have also examined the process of development and recovery of the tyrosinase isozymes in cells in which the interruption of melanogenesis has been released by the removal of these glycosylation inhibitors. The recovery process, which occurs during the period after interrupted melanogenesis and is a process of remelanization, has been biochemically followed. Tyrosinases obtained from the deoxycholate-solubilized large-granule fraction of these melanoma cells have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immediately after removal (0 h recovery) of the glycosylation inhibitor, loss of melanization and T3 is accompanied by T1 heterogeneity which is visualized as two electrophoretically distinct species, T1' and T1''. At this time, T1' and T1'' do not have a concanavalin A affinitive carbohydrate moiety but do possess in vitro dopa reactivity. When recovery of melanization begins visibly 24 h later, T3 is re-formed with disappearance of T1 heterogeneity. By 48 h, the previous normal level of melanization is almost attained. These results suggest that maturation of tyrosinase may occur via T1' and T1'' as precursors of T3, or possibly T1 through the addition of N-glycosydically linked oligosaccharide moieties which can be interrupted by glucosamine and tunicamycin.