IMMUNOCYTOCHEMICAL EVIDENCE FOR THE CYTOPLASMIC LOCALIZATION AND DIFFERENTIAL EXPRESSION DURING THE CELL-CYCLE OF THE M1-SUBUNIT AND M2-SUBUNIT OF MAMMALIAN RIBONUCLEOTIDE REDUCTASE
IMMUNOCYTOCHEMICAL EVIDENCE FOR THE CYTOPLASMIC LOCALIZATION AND DIFFERENTIAL EXPRESSION DURING THE CELL-CYCLE OF THE M1-SUBUNIT AND M2-SUBUNIT OF MAMMALIAN RIBONUCLEOTIDE REDUCTASE
复制标题
DOI：
10.1002/j.1460-2075.1988.tb02987.x
复制
发表时间：
1988-06-01
期刊：
影响因子：
11.4
通讯作者：
ROZELL, B
中科院分区：
文献类型：
--
作者：
ENGSTROM, Y;ROZELL, B
哺乳动物的核糖核苷酸还原酶由两个不同的亚基组成，即蛋白质M1和M2。我们制备并鉴定了针对小鼠核糖核苷酸还原酶M2蛋白的大鼠多克隆和单抗。利用这些抗体进行免疫细胞化学研究，在培养的亲代细胞和抗羟基脲的过量产生M2的小鼠TA3细胞中，以及在不同小鼠组织的细胞中，M2蛋白都被证明是专一的细胞质定位。这些数据，加上先前证明的M1亚基的细胞质定位，清楚地表明核糖核苷酸还原酶是一种细胞质酶。结合抗M2抗体和单抗M1抗体，可以对单个细胞中的这两个亚单位进行双标记免疫荧光研究。仅.apprx。在对数生长的培养中，50%的细胞含有免疫可检测到的蛋白M2，而所有细胞中都存在M1特异性染色。M2染色与S期细胞比例有很好的相关性。在组织中，仅有活跃分裂的细胞被两种抗体染色，且M2抗体染色的细胞总是比M1抗体染色的细胞少。因此，我们的数据为早期提出的核糖核苷酸还原酶M1和M2亚单位细胞周期中的差异调控模型提供了独立的证据。
Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2. We have produced and characterized rat polyclonal and monoclonal antibodies directed against protein M2 of mouse ribonucleotide reductase. Using these antibodies for immunocytochemical studies, an exclusively cytoplasmic localization of protein M2 was demonstrated both in cultured parent and hydroxyurea-resistant, M2-over-producing mouse TA3 cells, and in cells from various mouse tissues. These data, together with the previously demonstrated cytoplasmic localization of the M1 subunit, clearly show that ribonucleotide reductase is a cytoplasmic enzyme. Combining the anti-M2 antibodies with a monoclonal anti-M1 antibody allowed for double-labelling immunofluorescence studies of the two subunits in individual cells. Only .apprx. 50% of the cells in a logarithmically growing culture contained immunodetectable protein M2, while the M1-specific staining was present in all cells. The M2 staining correlates well with the proportion of cells in the S-phase of the cell cycle. In tissues, only actively dividing cells stained with either antibody and there were always fewer cells stained with the M2-antibodies than with the M1-antibody. Our data therefore present independent evidence for the earlier proposed model of a differential regulation during the cell cycle of the M1 and M2 subunits of ribonucleotide reductase.