Preparation of purified tyrosinase devoid of dopachrome tautomerase from mammalian malignant melanocytes.
Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60 degrees C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.
Valverde
P
P
Dpto. de Bioquimica, Facultad de Medicina, Universidad de Murcia, Spain.
Jiménez-Cervantes
C
C
Salinas
C
C
García-Borrón
J C
JC
Solano
F
F
Lozano
J A
JA
D000818:Animals; D016477:Artifacts; D002845:Chromatography; D003124:Colorimetry; D017886:Durapatite; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006358:Hot Temperature; D019746:Intramolecular Oxidoreductases; D007535:Isomerases; D008543:Melanins / Q000096:biosynthesis; D008544:Melanocytes / Q000201:enzymology*; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000302:isolation & purification*; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D008543:Melanins; D009363:Neoplasm Proteins; D017886:Durapatite; D014442:Monophenol Monooxygenase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase