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-relat ed protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblot ting and enzyme assays to copurify from extracts of Cloudman S91 melan oma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these prote ins. The tyrosine hydroxylase activity of tyrosinase was reduced in th e 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 extrac ts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight ''multimers'' of <similar to > 200 to > 700 kilodaltons. Extraction of cells with buffers containing the de tergent CHAPS preserved the high molecular weight multimers; Triton X- 100 caused their dissociation into monomers. Low pH, low ionic strengt h, and millimolar concentrations of calcium ions favored the maintenan ce of multimers. The results of this study demonstrate that the partic ipation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility tha t some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.