HIGH-MOLECULAR-WEIGHT FORMS OF TYROSINASE AND THE TYROSINASE-RELATED PROTEINS - EVIDENCE FOR A MELANOGENIC COMPLEX
HIGH-MOLECULAR-WEIGHT FORMS OF TYROSINASE AND THE TYROSINASE-RELATED PROTEINS - EVIDENCE FOR A MELANOGENIC COMPLEX
复制标题
DOI：
10.1111/1523-1747.ep12392743
复制
发表时间：
1994-08-01
影响因子：
6.5
通讯作者：
PAWELEK, JM
中科院分区：
文献类型：
--
作者：
ORLOW, SJ;ZHOU, BK;PAWELEK, JM
酪氨酸酶，酪氨酸酶相关蛋白-1（TRP-1）和酪氨酸酶相关蛋白-2（TRP-2，多巴色素互变异构酶）通过免疫印迹和酶测定显示从Cloudman S91黑色素瘤细胞提取物中共纯化。TRP-1和TRP-2的抗体免疫沉淀酪氨酸酶活性，表明这些蛋白质之间的稳定相互作用（复合物）。酪氨酸酶的酪氨酸羟化酶活性降低的复合形式，与Triton X-100的治疗解离的复合物和激活的酪氨酸酶存在于it. To进一步研究这个复杂的，我们采用蔗糖密度梯度离心的提取物从培养的小鼠黑素细胞。酪氨酸酶、TRP-1和TRP-2都存在于200至> 700千道尔顿的高分子量“多聚体”中。用含有洗涤剂CHAPS的缓冲液提取细胞保存了高分子量的多聚体; Triton X-100使其解离成单体。低pH值，低离子强度，和毫摩尔浓度的钙离子有利于维护的多聚体。这项研究的结果表明，酪氨酸酶，TRP-1，和TRP-2的参与在一个多聚体复合物可能有重要的生理后果，并提出了一些众所周知的毛色基因之间的相互作用的可能性，可以解释的基因产物之间的分子间相互作用。
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 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.