Isolation of human tyrosinase from cultured melanoma cells.
Tyrosinase was isolated from cultured melanoma cells using a procedure involving solubilization of the enzyme by means of Triton X-100, followed by different types of chromatography and tryptic digestion to make the enzyme soluble even in the absence of detergent. Starting with a membranous material containing 72 mg protein, 0.21 mg tyrosinase was obtained. The recovery of tyrosinase was 36% of the quantity found in the membranous starting material. In order to acquire a completely purified enzyme preparation suitable for amino acid sequence analysis, SDS-PAGE followed by blotting onto a polyvinylidene difluoride membrane was performed as a final step. The apparent molecular weight was found to be 66,000. Determination of the amino acids of the aminoterminal portion by automated Edman degradation showed the following sequence: His-Phe-Pro-Arg-Ala-X-Val-Ser-Ser-Lys-Asn-Leu-Met-Glu-Lys-Glu-X-X-Pro-Pr o-The enzyme purified has an amino acid sequence identical with that of human tyrosinase deduced from c-DNA by Kwon et al. Striking similarities between our amino acid sequence and that predicted by Yamamoto et al. from mouse tyrosinase c-DNA were also observed.
Wittbjer
A
A
Department of Pharmacology, University of Lund, Malmö, Sweden.
Dahlbäck
B
B
Odh
G
G
Rosengren
A M
AM
Rosengren
E
E
Rorsman
H
H
D000595:Amino Acid Sequence; D004156:Catechol Oxidase / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008546:Melanoma, Experimental / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000032:analysis / Q000302:isolation & purification*; D012878:Skin Neoplasms / Q000201:enzymology*; D014407:Tumor Cells, Cultured / Q000201:enzymology
D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase