Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma.
The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.
D000818:Animals; D004156:Catechol Oxidase / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D007527:Isoenzymes / Q000302:isolation & purification; D046911:Macromolecular Substances; D008297:Male; D008545:Melanoma / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D007527:Isoenzymes; D046911:Macromolecular Substances; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase