Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1973 Jul;115(1):18–22. doi: 10.1128/jb.115.1.18-22.1973

Properties of Ribonucleoside Diphosphate Reductase in Nucleotide-Permeable Cells

Huber R Warner 1
PMCID: PMC246205  PMID: 4146181

Abstract

Ribonucleoside diphosphate (RDP) reductase activity can be readily assayed in ether-treated Escherichia coli cells. The rate of cytidine 5′-diphosphate (CDP) reduction observed in ether-treated cells by using saturating substrate concentrations is about 25% of the rate of de novo deoxyribonucleotide synthesis required to account for in vivo deoxyribonucleic acid synthesis. Optimal activity is observed in the presence of magnesium ions and a positive effector. Adenosine 5′-triphosphate (ATP), deoxy ATP (dATP), and deoxythimidine triphosphate serve as positive effectors, and dATP also serves as a negative effector. These effects on the activity in ether-treated cells resemble those observed in vitro with highly purified enzyme. When the RDP reductase activity in these cells is assayed by using high specific activity 3H-CDP as substrate, even at nonsaturating substrate concentrations, the sensitivity of the assay is sufficient to make it useful for the assay of the low levels of reductase activity in cells not derepressed by thymine starvation or in cells containing mutationally altered RDP reductase. This assay is much easier to perform than the usual in vitro assay, since thioredoxin, thioredoxin reductase, and enzyme subunits B1 or B2 need not be first purified and added to the reaction mixtures.

Full text

PDF
18

Selected References

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

  1. Biswas C., Hardy J., Beck W. S. Release of repressor control of ribonucleotide reductase by thymine starvation. J Biol Chem. 1965 Sep;240(9):3631–3640. [PubMed] [Google Scholar]
  2. Brown N. C., Canellakis Z. N., Lundin B., Reichard P., Thelander L. Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins B1 and B2. Eur J Biochem. 1969 Jul;9(4):561–573. doi: 10.1111/j.1432-1033.1969.tb00646.x. [DOI] [PubMed] [Google Scholar]
  3. Brown N. C., Reichard P. Ribonucleoside diphosphate reductase. Formation of active and inactive complexes of proteins B1 and B2. J Mol Biol. 1969 Nov 28;46(1):25–38. doi: 10.1016/0022-2836(69)90055-2. [DOI] [PubMed] [Google Scholar]
  4. Fuchs J. A., Karlström H. O., Warner H. R., Reichard P. Defective gene product in dnaF mutant of Escherichia coli. Nat New Biol. 1972 Jul 19;238(81):69–71. doi: 10.1038/newbio238069a0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Larsson A., Reichard P. Enzymatic synthesis of deoxyribonucleotides. IX. Allosteric effects in the reduction of pyrimidine ribonucleotides by the ribonucleoside diphosphate reductase system of Escherichia coli. J Biol Chem. 1966 Jun 10;241(11):2533–2539. [PubMed] [Google Scholar]
  7. Larsson A., Reichard P. Enzymatic synthesis of deoxyribonucleotides. X. Reduction of purine ribonucleotides; allosteric behavior and substrate specificity of the enzyme system from Escherichia coli B. J Biol Chem. 1966 Jun 10;241(11):2540–2549. [PubMed] [Google Scholar]
  8. Moses R. E., Richardson C. C. Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc Natl Acad Sci U S A. 1970 Oct;67(2):674–681. doi: 10.1073/pnas.67.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. REICHARD P. Enzymatic synthesis of deoxyribonucleotides. I. Formation of deoxycytidine diphosphate from cytidine diphosphate with enzymes from Escherichia coli. J Biol Chem. 1962 Nov;237:3513–3519. [PubMed] [Google Scholar]
  10. STACEY K. A., SIMSON E. IMPROVED METHOD FOR THE ISOLATION OF THYMINE-REQUIRING MUTANTS OF ESCHERICHIA COLI. J Bacteriol. 1965 Aug;90:554–555. doi: 10.1128/jb.90.2.554-555.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Short E. V., Jr, Warner H. R., Koerner J. F. The effect of cupric ions on the indole reaction for the determination of deoxyribonucleic acid. J Biol Chem. 1968 Jun 25;243(12):3342–3344. [PubMed] [Google Scholar]
  12. Vosberg H. P., Hoffmann-Berling H. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J Mol Biol. 1971 Jun 28;58(3):739–753. doi: 10.1016/0022-2836(71)90037-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES