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. Copyright © 1978, Wiley Blackwell. All rights reserved
