Orlow S J, Zhou B K, Boissy R E, Pifko-Hirst S
Ronald O. Perelman Department of Dermatology, New York University School of Medicine, NY 10016.
J Invest Dermatol. 1993 Aug;101(2):141-4. doi: 10.1111/1523-1747.ep12363626.
Antiserum raised in rabbits against the Triton X-100 insoluble fraction of melanosomes from mouse melanoma cells specifically decorates the internal matrix of melanosomes in immunoelectron microscopy. In metabolic labeling studies, the antiserum recognizes a protein of 94 kDa, which is processed to a band of 53 kDa. Whereas the precursor is relatively soluble in buffers containing Triton X-100, the processed protein requires the addition of sodium dodecyl sulfate for effective solubilization, as would be expected for a melanosomal matrix constituent. Tunicamycin reduces the Mr of the nascent protein to 75 kDa, but deoxymannojirimycin and swainsonine have no effect, suggesting that following initial glycosylation in the endoplasmic reticulum, the protein is not subject to processing by glycosidases in the Golgi apparatus or may bypass it entirely. Subcellular fractionation followed by immunoblotting confirms that the protein is present in the melanosome-rich, large granule fraction. Expression of the protein is regulated differently from that of the tyrosinase-related protein family. Conditions that greatly stimulate expression of tyrosinase-related proteins do not affect matrix protein expression, nor is the protein immunologically related to the tyrosinase-related protein family. Our results suggest that we have identified an authentic component of the mammalian melanosomal matrix, and that its characteristics lend support to a bipartite pathway for melanosomal biogenesis.
用兔制备的抗小鼠黑色素瘤细胞黑素小体中Triton X - 100不溶部分的抗血清，在免疫电子显微镜下能特异性地标记黑素小体的内部基质。在代谢标记研究中，该抗血清识别一种94 kDa的蛋白质，它会被加工成一条53 kDa的条带。虽然前体在含有Triton X - 100的缓冲液中相对可溶，但加工后的蛋白质需要添加十二烷基硫酸钠才能有效溶解，这正如黑素小体基质成分所预期的那样。衣霉素将新生蛋白质的分子量降低到75 kDa，但脱氧甘露基野尻霉素和苦马豆素没有作用，这表明在内质网中进行初始糖基化后，该蛋白质不会受到高尔基体中糖苷酶的加工，或者可能完全绕过高尔基体。亚细胞分级分离后进行免疫印迹证实该蛋白质存在于富含黑素小体的大颗粒部分。该蛋白质的表达调控与酪氨酸酶相关蛋白家族不同。极大刺激酪氨酸酶相关蛋白表达的条件不会影响基质蛋白表达，并且该蛋白质与酪氨酸酶相关蛋白家族也没有免疫相关性。我们的结果表明，我们已经鉴定出哺乳动物黑素小体基质的一种真实成分，并且其特性支持黑素小体生物发生的二分途径。
