High-molecular-weight forms of tyrosinase and the tyrosinase-related proteins: evidence for a melanogenic complex.
Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.
Orlow
S J
SJ
Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York.
Zhou
B K
BK
Chakraborty
A K
AK
Drucker
M
M
Pifko-Hirst
S
S
Pawelek
J M
JM
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000032:analysis / Q000737:chemistry; D008544:Melanocytes / Q000737:chemistry / Q000201:enzymology; D008546:Melanoma, Experimental; D008562:Membrane Glycoproteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000032:analysis* / Q000737:chemistry; D017830:Octoxynol / Q000494:pharmacology; D009994:Osmolar Concentration; D010088:Oxidoreductases; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D013395:Sucrose; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D011506:Proteins; D013395:Sucrose; D017830:Octoxynol; D010088:Oxidoreductases; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase; D002118:Calcium