TYROSINASE ISOENZYMES IN MAMMALIAN MELANOCYTES .1. BIOCHEMICAL-CHARACTERIZATION OF 2 MELANOSOMAL TYROSINASES FROM B16 MOUSE MELANOMA

B-16 mouse melanoma melanosomes contain two forms of tyrosinase that c an 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 intraorgan ular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form o f lower electrophoretic mobility displayed a higher K(m) for 3,4-dihyd roxy-L-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lo wer ratio of tyrosine hydroxylation to Dopa oxidation. The form of hig her electrophoretic mobility displayed lower values of K(m) for both s ubstrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence Of L-dopa. Both forms were stereospec ific for the L isomers and sensitive to the specific tyrosinase inhibi tor 2-phenylthiourea. These forms do not appear to result from differe nt 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.