Tyrosinase isoenzymes in mammalian melanocytes. 2. Differential activation by alpha-melanocyte-stimulating hormone.
In mouse melanoma melanocytes, alpha-melanocyte-stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not 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 induction 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 appears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase activity of control and theophylline-treated extracts displayed several kinetic differences, including different Km values for both substrates and requirements for the cofactor L-dopa. SDS/PAGE, followed by a sensitive specific activity stain, demonstrated that melanosomes of control cells contain one lower-electrophoretic-mobility form of tyrosinase, whereas melanosomes of cells treated with either theophylline or MSH display, in addition to the lower-mobility form, a faster-migrating activity band. These tyrosinase forms are not interconvertible by proteolysis or deglycosylation. Their nature is discussed as related to the properties of the previously described low- and high-electrophoretic-mobility tyrosinases (LEMT and HEMT), as well as of the proteins encoded by the c and b loci.
Valverde
P
P
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain.
Garcia-Borron
J C
JC
Jimenez-Cervantes
C
C
Solano
F
F
Lozano
J A
JA
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004790:Enzyme Induction; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D008543:Melanins / Q000096:biosynthesis*; D009074:Melanocyte-Stimulating Hormones / Q000494:pharmacology*; D008544:Melanocytes / Q000187:drug effects / Q000201:enzymology*; D008546:Melanoma, Experimental; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D014442:Monophenol Monooxygenase / Q000096:biosynthesis / Q000378:metabolism*; D014176:Protein Biosynthesis; D013806:Theophylline / Q000494:pharmacology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D007527:Isoenzymes; D008543:Melanins; D009074:Melanocyte-Stimulating Hormones; D013806:Theophylline; D014446:Tyrosine 3-Monooxygenase; D014442:Monophenol Monooxygenase