Calreticulin-melatonin -: An unexpected relationship
Calreticulin-melatonin -: An unexpected relationship
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DOI：
10.1046/j.1432-1033.2003.03430.x
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发表时间：
2003-03-01
期刊：
影响因子：
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通讯作者：
Acuña-Castroviejo, D
中科院分区：
文献类型：
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作者：
Macías, M;Escames, G;Acuña-Castroviejo, D
越来越多的证据表明褪黑激素可以在核水平上发挥一些作用。先前使用结合技术的实验清楚地表明大鼠肝脏细胞核中存在特定的褪黑激素结合位点。为了进一步鉴定这些位点，用不同百分比的硫酸铵处理大鼠肝细胞的核提取物，并通过亲和层析纯化。随后的配体印迹分析显示存在大约 60 kDa 和大约 74 kDa 的两种与褪黑激素特异性结合的多肽。 N 端序列分析表明，60 kDa 蛋白与大鼠钙网蛋白具有高度同源性，而 74 kDa 蛋白与任何已知蛋白均无同源性。通过将 2-[(125) I]褪黑激素与重组钙网蛋白一起孵育，进一步表征了褪黑激素与钙网蛋白的结合。结合动力学显示 K (d) = 1.08 +/- 0.2 nm 和 B (max) = 290 +/- 34 fmol.mg 蛋白质(-1) ，与细胞中褪黑激素的其他结合位点兼容。通过蛋白质印迹分析进一步鉴定了钙网蛋白的存在，并通过蛋白质印迹和抗钙连接蛋白 Ig 免疫染色评估了我们的材料中不存在内质网污染。结果表明，钙网蛋白可能代表一类新的高亲和力褪黑激素结合位点，涉及吲哚胺的某些功能，包括基因组调节。
Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specific melatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts from rat hepatocytes were treated with different percentages of ammonium sulfate and purified by affinity chromatography. Subsequent ligand blot analysis shows the presence of two polypeptides of approximate to 60 and approximate to 74 kDa that bind specifically to melatonin. N-Terminal sequence analysis showed that the 60 kDa protein shares a high homology with rat calreticulin, whereas the 74 kDa protein shows no homology with any known protein. The binding of melatonin to calreticulin was further characterized incubating 2-[(125) I]melatonin with recombinant calreticulin. Binding kinetics show a K (d) = 1.08 +/- 0.2 nm and B (max) = 290 +/- 34 fmol.mg protein(-1) , compatible with other binding sites of melatonin in the cell. The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti-calnexin Ig. The results suggest that calreticulin may represent a new class of high-affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation.