Particulate Tyrosinase of Human Malignant Melanoma

Studies of mammalian tyrosinase have been conducted principally with soluble forms (T1 faster, T2 slower migrating by gel electrophoresis). However, a majority of tyrosinase from mouse and human melanoma tissue is of a particulate (T3) nature, being bound to melanosomes. In addition, T1 and T2 enzymes have been considered isozymes with T3 being a separate enzyme. Using human malignant melanoma tissue, solubilization and purification of particulate tyrosinase have been attempted. Melanosome-bound tyrosinase was solubilized with sodium cholate and purified 1500-fold by ammonium sulfate fractionation, DEAE-cellulose column chromatography, concanavalin A affinity chromatography, and Sephadex G-150 gel filtration. Tryptic cleavage of this solubilized tyrosinase yielded a fast migrating tyrosinase by gel electrophoresis. This trypsin-cleaved tyrosinase was purified by Sephadex G-150 and DEAE-cellulose columns with an apparent final yield of 56% and an approximate 10000-fold purification. This preparation gave a single band by 7% gel electrophoresis and 5% sodium dodecyl sulfate gel electrophoresis. In addition, electrophoresis indicated that this tyrosinase was a glycoprotein showing positive staining by periodic acid/Schiff stain. This enzyme oxidized both l-tyrosine and l-3,4-dihydroxyphenylalanine at a similar rate. The molecular weight was approximated at 66 700 by sodium dodecyl sulfate gel electrophoresis. This enzyme migrated to the T1 position by gel electrophoresis; however, upon treatment with neuraminidase, it shifted to the T2 position. Sialic acid content was determined as 4.8%. Copper content was estimated as 2 atoms/molecule. These results tend to support the dynamic conversion of T3 T1 T2 rather than their existence as separate enzymes.