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. 1979 Jun 25;6(8):2919–2928. doi: 10.1093/nar/6.8.2919

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.

J L Drocourt, D C Thang, R H Buckingham, M N Thang
PMCID: PMC327902  PMID: 379831

Abstract

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Deutscher M. P., Masiakowski P. Binding of tRNA nucleotidyltransferase to Affi-Gel Blue: rapid purification of the enzyme and binding studies. Nucleic Acids Res. 1978 Jun;5(6):1947–1954. doi: 10.1093/nar/5.6.1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Drocourt J. L., Thang D. C., Thang M. N. Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein. Eur J Biochem. 1978 Jan 16;82(2):355–362. doi: 10.1111/j.1432-1033.1978.tb12030.x. [DOI] [PubMed] [Google Scholar]
  3. Joseph D. R., Muench K. H. Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. I. Purification of the enzyme and of tryptrophan transfer ribonucleic acid. J Biol Chem. 1971 Dec 25;246(24):7602–7609. [PubMed] [Google Scholar]
  4. Krauss G., Riesner D., Maass G. Mechanism of tRNA-synthetase recognition: role of terminal A. Nucleic Acids Res. 1977 Jul;4(7):2253–2262. doi: 10.1093/nar/4.7.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Moe J. G., Piszkiewicz D. Purification of isoleucyl transfer ribonucleic acid synthetase by affinity chromatography on blue dextran-sepharose. FEBS Lett. 1976 Dec 15;72(1):147–150. doi: 10.1016/0014-5793(76)80832-0. [DOI] [PubMed] [Google Scholar]
  6. Rigler R., Pachmann U., Hirsch R., Zachau H. G. On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition. Eur J Biochem. 1976 May 17;65(1):307–315. doi: 10.1111/j.1432-1033.1976.tb10418.x. [DOI] [PubMed] [Google Scholar]
  7. Ryan L. D., Vestling C. S. Rapid purification of lactate dehydrogenase from rat liver and hepatoma: a new approach. Arch Biochem Biophys. 1974 Jan;160(1):279–284. doi: 10.1016/s0003-9861(74)80035-4. [DOI] [PubMed] [Google Scholar]
  8. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  9. Seno T., Kobayashi M., Nishimura S. Purification of Excherichia coli methionine tRNAF and methionine tRNAM and studies on their biophysical and biochemical properties. Biochim Biophys Acta. 1968 Nov 20;169(1):80–94. doi: 10.1016/0005-2787(68)90010-5. [DOI] [PubMed] [Google Scholar]
  10. Thang M. N., Harvey R. A., Grunberg-Manago M. Model for the elongation of polynucleotide chains by polynucleotide phosphorylase. J Mol Biol. 1970 Oct 28;53(2):261–280. doi: 10.1016/0022-2836(70)90299-8. [DOI] [PubMed] [Google Scholar]
  11. Thompson S. T., Cass K. H., Stellwagen E. Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins. Proc Natl Acad Sci U S A. 1975 Feb;72(2):669–672. doi: 10.1073/pnas.72.2.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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