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Alfred Kwan Daniel A. Kaplansky Martin Gross 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1984,783(1):80-88
We have separated and purified two forms of Met-tRNAf deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes. Deacylase I is a minor activity with an S20,w of 5.9, D20,w of 4.9 and Mr of 110 000, while deacylase II is the major activity with an S20,w of 3.3, D20,w of 7.1 and Mr of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [35S]Met-tRNAf to [35S]methionine and tRNAMetf and have no effect on reticulocyte [35S]fMet-tRNAf, [3H]Ala-tRNA or [3H]Lys-tRNA. However, while deacylase I has similar activity throughout the pH range of 6.1–8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1. In addition, deacylase II shows a much greater affinity for pure Met-tRNAf than deacylase I (Km of 1.5–3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNAMetf, deacylase I is inhibited similarly by any added tRNA. 相似文献
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Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases 总被引:4,自引:0,他引:4
R Petryshyn F Rosa R Fagard D Levin I M London 《Biochemical and biophysical research communications》1984,119(3):891-899
Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis. 相似文献
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Ray Petryshyn Daniel H. Levin Irving M. London 《Biochemical and biophysical research communications》1980,94(4):1190-1198
Double-stranded RNA (dsRNA) activates a cyclic 3′: 5′-AMP independent protein kinase (dsI) in reticulocyte lysates which inhibits protein synthesis by phosphorylating the 38, 000 dalton (38K) subunit of the initiation factor eIF-2 (eIF-2α). A latent precursor form of dsI (latent dsI) has been partially purified (1000–2000 fold) from lysates. Activation of dsI at all stages in the purification of latent dsI requires ATP and low levels of dsRNA (1–20 ng/ml), and is accompanied by the phosphorylation of a broad 67,000 dalton (67K) band. However, as purification proceeds the 67K band is resolved into two phosphorylated polypeptides of 68,500 and 67,000 daltons (). Although latent dsI and activated dsI have distinctly different chromatographic properties, both forms have similar molecular weights (~120,000) and similar sedimentation coefficients (~3.8S) in glycerol gradients. The data support the view that one or both components of the doublet are associated with the dsRNA-dependent protein kinase activity. 相似文献
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Ellen Z. Baum Vivian G. Ernst 《Biochemical and biophysical research communications》1983,114(1):41-49
Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A+) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes. 相似文献
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