Summary
The soluble and insoluble (particle-bound) tyrosinase (EC 1.10.3.1) present in extracts of Harding-Passey, B-16, and Cloudman S-91 mouse melanoma was examined by means of differential solubility, continuous-flow paper electrophoresis and acrylamide-gel electrophoresis.
Two soluble tyrosinases (T1 and T2) were found to exist as distinct entities in each type of melanoma examined. The relative concentration of these enzymes was not identical in the different types of melanoma, but their electrophoretic mobility was comparable in all three types of melanoma.
Insoluble tyrosinase (T3a) is solubilized (dissociated) in the presence of urea or a commercially available dispersing agent with solubilizing action similar to that of sodium dodecyl sulfate, but with special properties of its own. Tyrosinase that has been solubilized by this agent can be shown by acrylamide-gel electrophoresis to be composed of at least four separate enzymic moieties which appear as distinct bands in the gel. When the insoluble enzyme (T3a) is solubilized with urea, on the other hand, part of the enzyme is released in the supernatant solution in soluble form, T1. After continuous-flow paper electrophoresis, the tyrosinase solubilized by the dispersing agent exhibits unique electrophoretic mobility, thermostability, pH sensitivity, and thermodynamic constants of inactivation. Under appropriate conditions, conventional Michaelis-Menton kinetics are observable when l-tyrosine is used as a substrate.
The origin of T1, T2, and T3 within the melanocyte and the role of these enzymes in the formation of melanin are discussed.
This work was supported by Research Grants CA-08292 and CA-05010 from the NIH, USPHS, and the Damon Runyon Memorial Fund.
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