Tyrosinase isoenzymes: two melanosomal tyrosinases with different kinetic properties and susceptibility to inhibition by calcium.
Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS-PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and dopa oxidase activity and were stereospecific and sensitive to phenylthiourea, providing that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme. Immunoprecipitation and Western blots performed with the specific antibody alpha PEP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were noncompetitively inhibited by Ca2+ at physiologically relevant concentrations. However, the b protein was apparently more susceptible, since maximal inhibition was reached at lower Ca2+ concentrations for LEMT. Moreover, binding of Ca2+ to the tyrosinases resulted in a noticeable thermal destabilization of the enzymes, which was also more pronounced for LEMT.
Jiménez-Cervantes
C
C
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain.
Solano
F
F
Lozano
J A
JA
Garcia-Borrón
J C
JC
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002122:Calcium Chloride / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008544:Melanocytes / Q000201:enzymology*; D008546:Melanoma, Experimental / Q000201:enzymology; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D015636:Magnesium Chloride; D014442:Monophenol Monooxygenase; D002122:Calcium Chloride; D002118:Calcium