Tyrosinase isoenzymes in mammalian melanocytes. 1. Biochemical characterization of two melanosomal tyrosinases from B16 mouse melanoma.
B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Km for 3,4-dihydroxy-L-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of Km for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L-dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2-phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.
Jimenez-Cervantes
C
C
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain.
Garcia-Borron
J C
JC
Valverde
P
P
Solano
F
F
Lozano
J A
JA
D000818:Animals; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D008544:Melanocytes / Q000201:enzymology*; D008546:Melanoma, Experimental; D051379:Mice; D014442:Monophenol Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D044902:beta-Mannosidase
D007527:Isoenzymes; D014443:Tyrosine; D007980:Levodopa; D014442:Monophenol Monooxygenase; D008361:Mannosidases; D009439:Neuraminidase; D044902:beta-Mannosidase; D014357:Trypsin