Proteolysis with trypsin of mammalian tyrosinase isoforms from B16 mouse melanoma.
In spite of the central role of tyrosinase in mammalian pigmentation, few data are available on its structure and structure-function relationships based on direct analysis of the protein. A number of reasons have been invoked to account for this situation, including the problems for its purification and its resistance to proteases. However, no study on the effects of proteases on purified tyrosinase has been reported. We have purified the melanosomal and cytosolic tyrosinases from B16 mouse melanoma and analyzed their susceptibility to trypsin digestion. Both isoforms are sensitive to trypsin, and display similar peptide maps and kinetics of proteolysis, suggesting that they are products of the same gene. The peptide maps and the kinetics of appearance of the fragments were consistent with the sequential removal of N-terminal peptides, leading to a core of 55.3 kDa for the melanosomal form and 48.6 kDa for the cytosolic enzyme. This core was apparently resistant to further proteolysis and catalytically inactive. The difference in molecular weight for the core of the cytosolic and melanosomal forms is the same as that calculated for the native isoforms. The kinetics of enzyme inactivation indicate that the tyrosine hydroxylase and Dopa oxidase activities of tyrosinase are lost at the same rate, and should therefore display similar if not identical structural requirements. The results are discussed in terms of the relationship of both isoforms and of the putative protein sequences deduced from the cDNA clones proposed for tyrosinase.
Valverde
P
P
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Murcia.
García-Borrón
J C
JC
Solano
F
F
Lozano
J A
JA
D000818:Animals; D002458:Cell Fractionation; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008544:Melanocytes / Q000201:enzymology; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism*
D007527:Isoenzymes; D014442:Monophenol Monooxygenase; D014357:Trypsin