Abstract
A tyrosinase has been purified from the skin of the frog Xenopus laevis. Dihydroxyphenylalanine oxidase and tyrosine hydroxylase activities co-purify throughout the procedure. The enzyme is isolated in an inactive form, but both enzymatic activities are activated by a variety of anionic detergents. Of these, sodium dodecyl sulfate (NaDodSO4) is the most effective. The enzyme activation occurs at NaDodSO4 concentrations well below the critical micelle concentration and it remains active at concentrations as high as 30 mM (1%). Neither activity is stimulated by cationic or nonionic detergents, or a variety of other agents, including trypsin. The purified tyrosinase is a glycoprotein having a polypeptide Mr = 175,000 by NaDodSO4-polyacrylamide gel electrophoresis. This monomeric species is enzymatically active in the presence of NaDodSO4. Detergent-activated tyrosinase has a KM for dihydroxyphenylalanine of 6 X 10(-4) M and a KM for tyrosine of 4 X 10(-4) M. Both activities are inhibited by copper chelators but not by an iron chelator. Further characterization of the detergent activation of this enzyme is presented in a companion paper (Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 12542-12546).
Highlights
From the Biochemistry and Molecular Biology Section, Department of Biological Sciences,University Of California, Santa Barbara, California93106
The frog Xenopus laevis.Dihydroxyphenylalanine oxidase fact that fewof these studies havebeen carried out with and tyrosine hydroxylaseactivitiesco-purify through- purified enzyme has hindered the clarification of this quesout the procedure
The only case in which a purified form, but both enzymaticactivities are activated by a tyrosinase has been shown to be in a latent but activatable variety of anionic detergentsO. f these, sodium dodecyl form,is that of R a ~skinp(Bari~sasan~d Mc~Gu~e1,97%
Summary
We report the purification and partial characterization of activeat concentrationasshigh as 30 mM (1%)N. either a tyrosinase from the skin of the South African clawed frog, activity i s stimulated by cationic or nonionic deter- Xenopus k u i s. Both catalytic activities of the purified enpgTmggyeeelehaalpnnelettttaili-scedna,apceicotlnultrrMyieroviapafa,oihcvtfe=teoa6didrrv1eXiee7sttyiit5lsynyro.,ro0To-tosh‘0fhsie0niiMonsaitpbmashsaryseaeoneeNrnsdegoaahnalmDgyacKKeocseenodrM~otipSacsffrf0,sooN4poirtn-edearpcctiiDynoliheurloysydoddhaisSrncaion0rgvixy4eisnytl.oargepfDymnh4pezeiatsdynXei-enr-p-.opbztoklhyyehfnam-ooactwnoleimiptloahearpndiselsigeectiestynde,traalehoycstsiteisiwinrnvgeieaeseflslfunteenhatccssdet.aieaOvnfrievtrbshsateetaarriandtecddetatyeamicvtrtoeodaifrvntgaeaossedtttsnirhnabateysgytri,ocptaahnodrgonedeodtinfeetitiatndsoiszoeneayntsremos,er.fageWsan.ehdnTvoetawpOsrahhnicoeoottutswioy-r-. Tyrosinase (0-diphenokoxygen oxidoreductase; E1C.10.3.1) is a copper containing enzyme which catalyzes the two-step oxidation of tyrosine to melanin. This enzyme has been studied in a large variety of micro~al,plant, and animal systems (Nicolaus,1968).
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