Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins.
Within mammalian melanocytes, melanin biosynthesis is controlled by three enzymes structurally related: tyrosinase and two tyrosinase related proteins, TRP1 and TRP2. These melanosomal enzymes are integral membrane proteins with a carboxyl tail oriented to the cytoplasm, a single membrane-spanning helix and the bulk of the protein located inside the melanosome. Their solubilization is usually carried out by treatment of melanosomal preparations with non-ionic detergents, but, so far, no comparative study of the effect of the detergents employed on the properties of the solubilized proteins has been reported. We have compared the effect of the detergents Brij-35, Nonidet P-40, Tween-20, sodium deoxycholate and Triton X-114 on several properties of the melanogenic enzymes, including the solubilization yield, stability, electrophoretic behaviour and accessibility of epitopes located in the carboxyl tail to specific antibodies. Our data indicate that not only the total amount of enzymes solubilized, but also their relative proportions in the solubilized preparations depend on the detergent used. The non-ionic detergents apparently interact strongly with the melanogenic enzymes, affecting their mobility in SDS-PAGE, and might induce different conformations of the carboxyl tail. Complete replacement of lipids by the detergents results in a decreased stability that can be partially reversed by the addition of endogenous lipids. This treatment also produces a noticeable activation of the tyrosinase isoenzymes, which is higher for TRP1 than for tyrosinase. Taken together, these data show that the transmembrane and carboxyl fragments of the proteins of the tyrosinase family might modulate the stability and activity of the melanogenic enzymes.
Jiménez-Cervantes
C
C
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain.
García-Borrón
J C
JC
Lozano
J A
JA
Solano
F
F
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology*; D008055:Lipids / Q000494:pharmacology*; D008543:Melanins / Q000096:biosynthesis*; D008544:Melanocytes / Q000201:enzymology*; D008546:Melanoma, Experimental; D051379:Mice; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D017830:Octoxynol; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012995:Solubility; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D003902:Detergents; D008055:Lipids; D008543:Melanins; D000077423:Polidocanol; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D014446:Tyrosine 3-Monooxygenase; D014442:Monophenol Monooxygenase