TYROSINASE ISOENZYMES IN MAMMALIAN MELANOCYTES .2. DIFFERENTIAL ACTIVATION BY ALPHA-MELANOCYTE-STIMULATING HORMONE

In mouse melanoma melanocytes, alpha-melanocyte-stimulating hormone (M SH) stimulates differentiation, melanin synthesis and tyrosinase activ ity. However, the molecular mechanisms underlying these events have no t yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH , or its superpotent analog [Ahx4, DPhe7]MSH promotes a larger inducti on of tyrosine hydroxylase than of dopa oxidase activity in whole cell extracts. This higher activation of tyrosine hydroxylation was found not only in the melanosomal but also in the microsomal fraction; it ap pears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase ac tivity of control and theophylline-treated extracts displayed several kinetic differences, including different K(m) values for both substrat es and requirements for the cofactor L-dopa. SDS/PAGE, followed by a s ensitive specific activity stain, demonstrated that melanosomes of con trol cells contain one lower-electrophoretic-mobility form of tyrosina se, whereas melanosomes of cells treated with either theophylline or M SH display, in addition to the lower-mobility form, a faster-migrating activity band. These tyrosinase forms are not interconvertible by pro teolysis or deglycosylation. Their nature is discussed as related to t he properties of the previously described low- and high-electrophoreti c-mobility tyrosinases (LEMT and HEMT), as well as of the proteins enc oded by the c and b loci.