IDENTIFICATION OF NUCLEOLIN AS A BINDING-PROTEIN FOR MIDKINE (MK) AND HEPARIN-BINDING GROWTH-ASSOCIATED MOLECULE (HB-GAM)
IDENTIFICATION OF NUCLEOLIN AS A BINDING-PROTEIN FOR MIDKINE (MK) AND HEPARIN-BINDING GROWTH-ASSOCIATED MOLECULE (HB-GAM)
复制标题
DOI：
10.1093/oxfordjournals.jbchem.a124628
复制
发表时间：
1994-11-01
影响因子：
2.7
通讯作者：
MURAMATSU, T
中科院分区：
文献类型：
--
作者：
TAKE, M;TSUTSUI, J;MURAMATSU, T
中期因子（MK）是一种肝素结合生长/分化因子，分子量为13 kDa，结构上与成纤维细胞生长因子（FGF）无关。为了阐明MK的作用机制，我们对MK结合蛋白进行了研究。通过配体印迹实验，在PYS-2、3 T3和L细胞中鉴定出100-kDa蛋白质作为MK结合蛋白。该MK结合蛋白通过MK-琼脂糖柱上的亲和层析，随后通过SDS聚丙烯酰胺凝胶电泳进行纯化。N-末端23个氨基酸残基的序列测定表明，MK结合蛋白是核仁素，一种主要的核仁蛋白，其功能是作为细胞核和细胞质之间的穿梭蛋白，也位于细胞表面。肝素结合生长相关分子（HB-GAM）与MK具有50%的序列同一性，与麦芽糖结合蛋白融合，也与核仁素结合。另一方面，碱性成纤维细胞生长因子（bFGF）几乎没有绑定到核仁素在肝素的情况下，而MK和碱性成纤维细胞生长因子结合弱核仁素在肝素的存在下。免疫组化染色显示MK在血管瘤细胞核内定位。这些发现支持了MK和HB-GAM的一部分在与核仁素结合后易位到细胞核的假设。
Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK. A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.