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From wheat embryos, tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated. By chromatography on Sepharose 6B, DEAE-cellulose and affinity chromatography on tRNA-hydrazyl-Sepharose 4B, 7000-fold purification of the enzyme was achieved. The enzyme required for its activity Mg2+ or Mn2+ ion. ATP inhibited incorporation of CMP from CTP into lupin tRNA, and CTP acted as a competitive inhibitor of AMP incorporation from ATP. The regulatory role of ATP in incorporation of terminal CMP into tRNA is discussed. The incorporation of terminal CMP into tRNA deprived of terminal CCA or CA, was also studied. 相似文献
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The methylation of transfer RNA by methyl cobamide 总被引:3,自引:0,他引:3
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Hageman J van Waarde MA Zylicz A Walerych D Kampinga HH 《The Biochemical journal》2011,435(1):127-142
Humans contain many HSP (heat-shock protein) 70/HSPA- and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected, with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress. 相似文献
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Hsp90 chaperones wild-type p53 tumor suppressor protein 总被引:6,自引:0,他引:6
Walerych D Kudla G Gutkowska M Wawrzynow B Muller L King FW Helwak A Boros J Zylicz A Zylicz M 《The Journal of biological chemistry》2004,279(47):48836-48845
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Müller L Schaupp A Walerych D Wegele H Buchner J 《The Journal of biological chemistry》2004,279(47):48846-48854
The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation. 相似文献
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1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K12 and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[58Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active. 相似文献