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. 1972 Feb;109(2):584–593. doi: 10.1128/jb.109.2.584-593.1972

Leucyl-Transfer Ribonucleic Acid Synthetase from a Wild-Type and Temperature-Sensitive Mutant of Salmonella typhimurium1

T W Mikulka a,2, B I Stieglitz a,3, J M Calvo a
PMCID: PMC285181  PMID: 4550813

Abstract

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following Km values: leucine, 1.1 × 10−5m; adenosine triphosphate, 6.5 × 10−4m; tRNAILeu, 4.1 × 10−8m; tRNAIILeu, 4.3 × 10−8m; tRNAIIILeu, 5.3 × 10−8m; and tRNAIVLeu, 2.9 × 10−8m. The tRNALeu fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min−1. Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.

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Selected References

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

  1. Alexander R. R., Calvo J. M., Freundlich M. Mutants of Salmonella typhimurium with an altered leucyl-transfer ribonucleic acid synthetase. J Bacteriol. 1971 Apr;106(1):213–220. doi: 10.1128/jb.106.1.213-220.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett T. P. Evidence for one leucyl transfer ribonucleic acid synthetase with specificity for leucine transfer ribonucleic acids with different coding characteristics. J Biol Chem. 1969 Jun 25;244(12):3182–3187. [PubMed] [Google Scholar]
  3. Burns R. O., Calvo J., Margolin P., Umbarger H. E. Expression of the leucine operon. J Bacteriol. 1966 Apr;91(4):1570–1576. doi: 10.1128/jb.91.4.1570-1576.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Calvo J. M., Freundlich M., Umbarger H. E. Regulation of branched-chain amino acid biosynthesis in Salmonella typhimurium: isolation of regulatory mutants. J Bacteriol. 1969 Mar;97(3):1272–1282. doi: 10.1128/jb.97.3.1272-1282.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DIXON M. A nomogram for ammonium sulphate solutions. Biochem J. 1953 Jun;54(3):457–458. doi: 10.1042/bj0540457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gillam I., Millward S., Blew D., von Tigerstrom M., Wimmer E., Tener G. M. The separation of soluble ribonucleic acids on benzoylated diethylaminoethylcellulose. Biochemistry. 1967 Oct;6(10):3043–3056. doi: 10.1021/bi00862a011. [DOI] [PubMed] [Google Scholar]
  7. Hayashi H., Knowles J. R., Katze J. R., Lapointe J., Söll D. Purification of leucyl transfer ribonucleic acid synthetase from Escherichia coli. J Biol Chem. 1970 Mar 25;245(6):1401–1406. [PubMed] [Google Scholar]
  8. Kan J., Nirenberg M. W., Sueooka N. Coding specificity of Escherichia coli leucine transfer ribonucleic acids and effect of bacteriophage T2 infection. J Mol Biol. 1970 Sep 14;52(2):179–193. doi: 10.1016/0022-2836(70)90024-0. [DOI] [PubMed] [Google Scholar]
  9. Kan J., Sueoka N. Further evidence for a single leucyl transfer ribonucleic acid synthetase capable of charging five leucine transfer ribonucleic acids in Escherichia coli. J Biol Chem. 1971 Apr 10;246(7):2207–2210. [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. MARGOLIN P. Genetic fine structure of the leucine operon in Salmonella. Genetics. 1963 Mar;48:441–457. doi: 10.1093/genetics/48.3.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Neidhardt F. C. Roles of amino acid activating enzymes in cellular physiology. Bacteriol Rev. 1966 Dec;30(4):701–719. doi: 10.1128/br.30.4.701-719.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. RAMAKRISHNAN T., ADELBERG E. A. REGULATORY MECHANISMS IN THE BIOSYNTHESIS OF ISOLEUCINE AND VALINE. II. IDENTIFICATION OF TWO OPERATOR GENES. J Bacteriol. 1965 Mar;89:654–660. doi: 10.1128/jb.89.3.654-660.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ray D. K., Rappaport H. P. Further investigations of the multiple fractions of leucyl transfer ribonucleic acid synthetase activity from Escherichia coli. Biochim Biophys Acta. 1970 Jan 21;199(1):79–85. doi: 10.1016/0005-2787(70)90696-9. [DOI] [PubMed] [Google Scholar]
  15. Rouget P., Chapeville F. Leucyl-tRNA synthetase. Purification of two interconvertible forms and evidence for an interconversion factor. Eur J Biochem. 1970 Jul;14(3):498–508. doi: 10.1111/j.1432-1033.1970.tb00316.x. [DOI] [PubMed] [Google Scholar]
  16. Roy K. L., Söll D. Purification of five serine transfer ribonucleic acid species from Escherichia coli and their acylation by homologous and heterologous seryl transfer ribonucleic acid synthetases. J Biol Chem. 1970 Mar 25;245(6):1394–1400. [PubMed] [Google Scholar]
  17. Seifert W., Nass G., Zillig W. Electrophoretic separation of tRNA-bound leucyl-tRNA synthetase from Escherichia coli extracts. J Mol Biol. 1968 Apr 28;33(2):507–511. doi: 10.1016/0022-2836(68)90208-8. [DOI] [PubMed] [Google Scholar]
  18. Silbert D. F., Fink G. R., Ames B. N. Histidine regulatory mutants in Salmonella typhimurium 3. A class of regulatory mutants deficient in tRNA for histidine. J Mol Biol. 1966 Dec 28;22(2):335–347. doi: 10.1016/0022-2836(66)90136-7. [DOI] [PubMed] [Google Scholar]
  19. Smith H. O., Levine M. A phage P22 gene controlling integration of prophage. Virology. 1967 Feb;31(2):207–216. doi: 10.1016/0042-6822(67)90164-x. [DOI] [PubMed] [Google Scholar]
  20. Stieglitz B., Calvo J. M. Effect of 4-azaleucine upon leucine metabolism in Salmonella typhimurium. J Bacteriol. 1971 Oct;108(1):95–104. doi: 10.1128/jb.108.1.95-104.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vanhumbeeck J., Lurquin P. Purification and some properties of leucyl-tRNA synthetase from Bacillus stearothermophilus. Eur J Biochem. 1969 Sep;10(2):213–218. doi: 10.1111/j.1432-1033.1969.tb00676.x. [DOI] [PubMed] [Google Scholar]
  22. Weiss J. F., Kelmers A. D. A new chromatographic system for increased resolution of transfer ribonucleic acids. Biochemistry. 1967 Aug;6(8):2507–2513. doi: 10.1021/bi00860a030. [DOI] [PubMed] [Google Scholar]
  23. Yu C. T. Multiple forms of leucyl sRNA synthetase of E. coli. Cold Spring Harb Symp Quant Biol. 1966;31:565–570. doi: 10.1101/sqb.1966.031.01.073. [DOI] [PubMed] [Google Scholar]
  24. Yu C. T., Rappaport H. P. Multiple fractions of leucyl-transfer ribonucleic acid synthetase activity from Escherichia coli. Biochim Biophys Acta. 1966 Jul 20;123(1):134–141. doi: 10.1016/0005-2787(66)90166-3. [DOI] [PubMed] [Google Scholar]

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