C. Jimenezcervantes et al., TYROSINASE ISOENZYMES - 2 MELANOSOMAL TYROSINASES WITH DIFFERENT KINETIC-PROPERTIES AND SUSCEPTIBILITY TO INHIBITION BY CALCIUM, Pigment cell research, 7(5), 1994, pp. 291-297
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.