Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.
Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.
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7-甲基鸟苷 5-三磷酸的苯基叠氮化物取代和二苯甲酮取代的膦酰胺作为蛋白质合成起始因子 eIF-4E 和含有帽结合位点的蛋白水解片段的光亲和探针。
DOI：
10.1021/bi00475a016
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Rhoads,RE
中科院分区：
文献类型：
--
作者：
Chavan,AJ;Rychlik,W;Blaas,D;Kuechler,E;Watt,DS;Rhoads,RE
1990年1月17日收到的修订版Mandarin摘要：使用真核mRNA的含7-甲基鸟苷帽的三种光敏衍生物研究人红细胞和兔网织红细胞的蛋白质合成起始因子eIF-4 E。用先前描述的探针[7- 32 P]-Y-[[（4-苯甲酰基苯基）甲基]酰氨基]-7-甲基-GTP观察到eIF-4 E的灵敏和特异性标记[Blaas et al.（1982）Virology 116，339;缩写为[32 P] BPM]。合成第二种探针，其为m7 GTP的叠氮基苯基酪氨酸衍生物（[125 I] APTM），m7 GDP与[125 I]-A-（4-叠氮基苯基）-2-（磷酰胺基）-3-（4-羟基-3-碘苯基）丙酰胺的单酐。该探针允许在合成的最后一步而不是第一步快速和定量引入放射性，并将放射性标记置于弱PN键的蛋白质近端侧。测定[125 I] APTM的解离常数为6.9 pM，与m7 GTP的已发表值相当。m7 GTP和APTM作为用[125 I] APTM标记的eIF-4 E的竞争性抑制剂同样有效。与[32 P] BPM一样，[125 I] APTM标记全长（25 kDa）多肽和16 kDa降解产物（命名为eIF-4 E *），标记与存在的每种多肽的量成比例。还合成了第三种探针，m7 GTP的叠氮基苯基甘氨酸衍生物（[32 P] APGM），m7 GDP与[32 P]-A-（4-叠氮基苯基）-2-（磷酰胺基）乙酰胺的单酐，并显示特异性标记eIF-4 E。然而，与[32 P] BPM和[125 I] APTM不同，[32 P] APGM标记eIF-4 E * 比完整eIF-4 E容易约4倍。胰蛋白酶和CNBr裂解表明，eIF-4 E * 由eIF-4 E的蛋白酶抗性核心组成，该核心保留帽结合位点，并由大约1.5组成。mRNA进入多肽链起始循环涉及25-kDa多肽，真核起始因子（eIF）1 4 E识别含7-甲基鸟苷的帽[Rhoads（1988）和Sonenberg（1988）综述]。在动物细胞中，eIF-4 E以游离形式分离（Sonenberg等人，1979; Hellmann等人，1982）和与其它多肽的复合物，其中一种称为eIF-4F（Tahara等，1981; Grifo等人，1983; Edery等人，1983年）。不清楚mRNA的初始识别是通过游离eIF-4 E还是通过eIF-4F（Hiremath等人，1989年）。基于帽结构的化学探针已用于阐明eIF-4多肽组的功能和作用机制。Sonenberg和Shatkin（1977）使用高碘酸盐氧化、帽标记的mRNA，发现小麦胚芽线粒体后上清液或粗网织红细胞起始因子中的几种多肽在用NaBH 3CN还原后被标记。随后，Sonenberg（1981）表明，在不存在ATP的情况下，只有eIF-4 E与该探针交联，而46-kDa起始因子eIF-4A在存在ATP的情况下也交联。通过在帽中引入~（32）P代替~（3 H），大大提高了这种探针的灵敏度
Revised Manuscript Received January 17, 1990 abstract: Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe,[7-32P]-Y-[[(4-benzoylphenyl) methyl] amido]-7-methyl-GTP [Blaas et al.(1982) Virology 116, 339; abbreviated [32P] BPM], A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP ([125I] APTM), the monoanhydride of m7GDP with [125I]-A-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodophenyl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak PN bond. A dissociation constant of 6.9 pM was determined for [125I] APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I] APTM. Like [32P] BPM,[125I] APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP ([32P] APGM), the monoanhydride of m7GDP with [32P]-A-(4-azidophenyl)-2-(phosphoramido) acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P] BPMand [125I] APTM, however,[32P] APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximatelyThe entry of mRNA into the cycle of polypeptide chain initiation involves the recognition of the 7-methylguanosinecontaining cap by a 25-kDa polypeptide, eukaryotic initiation factor (elF) 1 4E [reviewed in Rhoads (1988) and Sonenberg (1988)]. In animal cells, eIF-4E is isolated both in free form (Sonenberg et al., 1979; Hellmann et al., 1982) and in com-plexes with otherpolypeptides, one of which is termed eIF-4F (Tahara et al., 1981; Grifo et al., 1983; Edery et al., 1983). It is not clear whether initial recognition of mRNA is by free eIF-4E or by eIF-4F (Hiremath et al., 1989). Chemical probes based on the cap structure have been useful in the elucidation of the function and mechanism of action of the eIF-4 group of polypeptides. Sonenberg and Shatkin (1977) used periodate-oxidized, cap-labeled mRNA and showed that several polypeptides in wheat germ postmito-chondrial supernate or crude reticulocyte initiation factors were labeled following reduction with NaBH3CN. Subsequently, Sonenberg (1981) showed that whereas only eIF-4E was cross-linked to this probe in the absence of ATP, the 46-kDa initiation factor eIF-4A was also cross-linked in the presence of ATP. The sensitivity of this type of probe was greatly enhanced by the introduction of 32P instead of 3H into thecap