PURIFICATION AND CHARACTERIZATION OF EUKARYOTIC TRANSLATIONAL INITIATION-FACTOR EIF-2B FROM LIVER
PURIFICATION AND CHARACTERIZATION OF EUKARYOTIC TRANSLATIONAL INITIATION-FACTOR EIF-2B FROM LIVER
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DOI：
10.1016/0304-4165(94)90079-5
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发表时间：
1994-12-15
影响因子：
3
通讯作者：
JEFFERSON, LS
中科院分区：
文献类型：
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作者：
KIMBALL, SR;KARINCH, AM;JEFFERSON, LS
真核起始因子（eIF）-2B纯化大于95%的同质性从大鼠和牛的肝脏。纯化的蛋白质由5个不同的亚基组成，表观分子量范围为30.9至89.1 kDa。全蛋白的特征在于它的斯托克斯半径和摩擦系数。发现β-、γ-和ε-亚基的等电点分别为6.4、6.9和约6.0; α-和δ-亚基没有很好地聚焦，因为这两种蛋白质的cDNA核苷酸序列预测的它们的等电点大于8.5。纯化的蛋白质用作抗原以产生针对ε-亚基的单克隆抗体。然后使用eIF-2B β单克隆抗体和针对eIF-2 α亚基的单克隆抗体直接定量大鼠肝脏和大鼠网织红细胞中eIF-2B和eIF-2的量。发现eIF-2B与eIF-2的比率约为100%。0.6在肝脏和网织红细胞中分别为0.3，这支持了这样的论点：仅部分总细胞eIF-2的磷酸化可能会将所有eIF-2B隔离为无活性的eIF-2 eIF-2B复合物。纯化的蛋白质也用作蛋白激酶测定中的底物。大鼠肝脏提取物显示含有针对ε-亚基的蛋白激酶活性，但没有eIF-2B的其他亚基。总的来说，这里介绍的研究是第一个显示eIF-2和eIF-2B在不同组织中的直接定量。它们还提供了证据，证明eIF-2B的ε-亚基是大鼠肝脏提取物中存在的唯一被蛋白激酶磷酸化的eIF-2B亚基。
Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein consisted of five nonidentical subunits with apparent molecular weights ranging from 30.9 to 89.1 kDa. The holoprotein was characterized in terms of its Stokes radius and frictional coefficient. The isoelectric points for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximate to 6.0, respectively; the alpha- and delta-subunits did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater than 8.5. The purified protein was used as antigen to generate monoclonal antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal antibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were then used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found to be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, supporting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2 eIF-2B complex. The purified protein was also used as substrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here are the first to show a direct quantitation of eIF-2 and eIF-2B in different tissues. They also provide evidence that the epsilon-subunit of eIF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.