Purification of tyrosinase to homogeneity based on its resistance to sodium dodecyl sulfate-proteinase K digestion.
A two-step method was used to purify enzymatically active tyrosinase to apparent homogeneity from B16/C3 mouse melanoma cells. The purification was based on our finding that tyrosinase was highly resistant to sodium dodecyl sulfate-proteinase K digestion. Following treatment with proteinase K, tyrosinase was separated from all detectable contaminants using DEAE-52 ion-exchange chromatography. Using this procedure, tyrosinase was purified to high specific activity and in yields greater than 50%. The purified enzyme was then used to generate high titers of specific polyclonal antibodies in rabbits. These antibodies were used to immunoprecipitate all tyrosinase isozymes from murine melanoma tumor extracts and cultured B16/C3 cells metabolically labeled with [35S]methionine.
Yurkow
E J
EJ
Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
Laskin
J D
JD
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004156:Catechol Oxidase / Q000302:isolation & purification*; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D007700:Kinetics; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000302:isolation & purification* / Q000378:metabolism; D012697:Serine Endopeptidases; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*
D000596:Amino Acids; D012967:Sodium Dodecyl Sulfate; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D012697:Serine Endopeptidases; D019286:Endopeptidase K