TYROSINASE ISOENZYMES - 2 MELANOSOMAL TYROSINASES WITH DIFFERENT KINETIC-PROPERTIES AND SUSCEPTIBILITY TO INHIBITION BY CALCIUM

Two forms of tyrosinase from B16 mouse melanoma were identified by non reducing SDS-PAGE after solubilization of crude melanosomal preparatio ns with the nonionic detergent Brij 35, These forms, named LEMT and HE MT (low and high electrophoretic mobility tyrosinase, respectively), w ere purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and d opa oxidase activity and were stereospecific and sensitive to phenylth iourea, proving that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme. Immunoprecip itation and Western blots performed with the specific antibody alpha P EP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were nonc ompetitively inhibited by Ca2+ at physiologically relevant concentrati ons. However, the b protein was apparently more susceptible, since max imal inhibition was reached at lower Ca2+ concentrations for LEMT. Mor eover, binding of Ca2+ to the tyrosinases resulted in a noticeable the rmal destabilization of the enzymes, which was also more pronounced fo r LEMT.