Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.
Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.
复制标题
兔网织红细胞裂解物中蛋白质合成的调节：血红素可逆翻译抑制剂的纯化和表征。
DOI：
10.1073/pnas.75.8.3654
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发表时间：
1978
影响因子：
11.1
通讯作者：
I. London
中科院分区：
文献类型：
--
作者：
H. Trachsel;R. S. Ranu;I. London
为了确定在血红素缺乏时激活并抑制蛋白质合成起始的蛋白激酶的调节机制，我们从兔网织红细胞中分离并纯化了血红素可逆形式的蛋白激酶。在非变性条件下，聚丙烯酰胺凝胶电泳后发现其抑制活性为单带。它以95000道尔顿多肽的形式在15%十二烷基硫酸钠/聚丙烯酰胺凝胶中迁移。这种纯化的抑制剂在ATP存在下自我磷酸化；磷酸化蛋白和抑制活性共同作用。该抑制剂在网状细胞裂解物中产生典型的双相抑制动力学，并磷酸化真核起始因子2 （eIF-2）的38,000道尔顿亚基；加入eIF-2后，抑制作用被逆转。与血红素不可逆抑制剂相反，该血红素可逆抑制剂在与20微米血红素孵育后不再具有抑制作用。与血红蛋白孵育也抑制自身磷酸化。与n -乙基马来酰亚胺处理一样，血红素可逆抑制剂在ATP存在下的预孵育增强了随后孵育中蛋白质合成的抑制作用。血红素可逆抑制剂的磷酸化和由于eIF-2的磷酸化而抑制裂解物中的蛋白质合成似乎是相关的。这些发现表明血红素直接作用于血红素可逆抑制剂。
To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.