INITIATION OF MAMMALIAN PROTEIN-SYNTHESIS .1. PURIFICATION AND CHARACTERIZATION OF 7 INITIATION-FACTORS
INITIATION OF MAMMALIAN PROTEIN-SYNTHESIS .1. PURIFICATION AND CHARACTERIZATION OF 7 INITIATION-FACTORS
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DOI：
10.1016/0022-2836(77)90268-6
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发表时间：
1977-01-01
影响因子：
5.6
通讯作者：
STAEHELIN, T
中科院分区：
文献类型：
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作者：
SCHREIER, MH;ERNI, B;STAEHELIN, T
从兔网织红细胞中纯化出蛋白质因子（7），推测所有这些因子都参与哺乳动物蛋白质合成的起始。它们被称为eIF-1、eIF-2、eIF-3、eIF-4A、eIF-4 B、eIF-4C和e-IF-5。从KCl中纯化粗核糖体包括用硫酸铵分级分离、离子交换色谱和按大小分离。起始因子的操作定义是其在高度纯化和分级系统中使用完全确定的延伸组分（即氨酰-tRNA、2种延伸因子EF-1和EF-2以及GTP）翻译天然信使RNA（珠蛋白mRNA）的要求。根据相同的标准，ATP被证明是启动所需的。除eIF-4 B（纯度为60-70%）外，将起始因子纯化至均质。他们的特点是物理蔗糖梯度离心和聚丙烯酰胺凝胶电泳在十二烷基硫酸钠的存在下。除eIF-2和eIF-3外，它们由MW范围为15，000（eIF-1）至约160，000（eIF-5）的单链多肽组成。因子eIF-2具有约35，000、50，000和55，000 MW的3个亚基。通过非解离条件下的凝胶电泳和沉降分析判断，因子eIF-3似乎是均一的。在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳显示至少9个亚基，分子量范围为约35，000 - 160，000。在蔗糖梯度上分离在7种起始因子、ATP和GTP存在下制备的起始复合物（mRNA.cntdot.Met-tRNAf.cntdot.80 S [Svedberg]核糖体），并显示当提供氨酰-tRNA、2种延伸因子和GTP时，其在多肽链延伸中具有完全活性。
Protein factors (7) were purified from rabbit reticulocytes, all of which are presumed to be involved in initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIf-4B, eIF-4C and e-IF-5. The purification from the KCl was of crude ribosomes involves fractionation with amononium sulfate, ion-exchange chromatography and separation by size. The operational definitition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the 2 elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was shown to be required for initiation. Initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60-70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in MW from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has 3 subunits of about 35,000, 50,000 and 55,000 MW. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate reveals at least 9 subunits ranging in MW from about 35,000-160,000. Initiation complexes (mRNA.cntdot.Met-tRNAf.cntdot.80 S [Svedberg] ribosome), made in the presence of the 7 initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the 2 elongation factors and GTP.