Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1976 Mar 1;153(3):533–541. doi: 10.1042/bj1530533

Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12.

R A Clegg
PMCID: PMC1172619  PMID: 782444

Abstract

1. Nitrate reductase was purified 134-fold from Escherichia coli K12. The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope. i. The purified enzyme exists in aqueous solution either as a monomer (mol. wt. about 220 000) or as an associated form (probably a tetramer; mol.wt. about 880 000). 3. The purified enzyme has three subunits with apparent mol.wts. of 150 000, 67000 and 65000. An additional subunit of apparent mol.wt. 20000 is present in a haem-containing fraction that is also produced by the preparative procedure described. 4. None of the enzyme subunits is present in the cell envelope of cells grown in the absence of nitrate. 5. Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3-activity.

Full text

PDF
533

Images in this article

536-1PLATE 1
on p.536-1

Selected References

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

  1. Clegg R. A., Skyrme J. E. The effects of iron-limited growth on the reduced nicotinamide-adenine dinucleotide dehydrogenase activity and the membrane proteins of Candida utilis mitochondria. Biochem J. 1973 Dec;136(4):1029–1037. doi: 10.1042/bj1361029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clegg R. A. The nitrate respiration complex of Escherichia coli: subunit composition. Biochem Soc Trans. 1975;3(5):689–691. doi: 10.1042/bst0030689. [DOI] [PubMed] [Google Scholar]
  3. Clegg R. A. The size of nitrate reductase in Escherichia coli. Biochem Soc Trans. 1975;3(5):691–694. doi: 10.1042/bst0030691. [DOI] [PubMed] [Google Scholar]
  4. Cohen P. The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions. Eur J Biochem. 1973 Apr 2;34(1):1–14. doi: 10.1111/j.1432-1033.1973.tb02721.x. [DOI] [PubMed] [Google Scholar]
  5. Enoch H. G., Lester R. L. The role of a novel cytochrome b-containing nitrate reductase and quinone in the in vitro reconstruction of formate-nitrate reductase activity of E. coli. Biochem Biophys Res Commun. 1974 Dec 23;61(4):1234–1241. doi: 10.1016/s0006-291x(74)80416-x. [DOI] [PubMed] [Google Scholar]
  6. Forget P. The bacterial nitrate reductases. Solubilization, purification and properties of the enzyme A of Escherichia coli K 12. Eur J Biochem. 1974 Mar 1;42(2):325–332. doi: 10.1111/j.1432-1033.1974.tb03343.x. [DOI] [PubMed] [Google Scholar]
  7. Inouye M., Yee M. L. Homogeneity of envelope proteins of Escherichia coli separated by gel electrophoresis in sodium dodecyl sulfate. J Bacteriol. 1973 Jan;113(1):304–312. doi: 10.1128/jb.113.1.304-312.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  10. MacGregor C. H., Schnaitman C. A. Alterations in the cytoplasmic membrane proteins of various chlorate-resistant mutants of Escherichia coli. J Bacteriol. 1971 Oct;108(1):564–570. doi: 10.1128/jb.108.1.564-570.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. MacGregor C. H., Schnaitman C. A., Normansell D. E. Purification and properties of nitrate reductase from Escherichia coli K12. J Biol Chem. 1974 Aug 25;249(16):5321–5327. [PubMed] [Google Scholar]
  12. MacGregor C. H. Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease. J Bacteriol. 1975 Mar;121(3):1102–1110. doi: 10.1128/jb.121.3.1102-1110.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  14. Ruiz-Herrera J., DeMoss J. A. Nitrate reductase complex of Escherichia coli K-12: participation of specific formate dehydrogenase and cytochrome b1 components in nitrate reduction. J Bacteriol. 1969 Sep;99(3):720–729. doi: 10.1128/jb.99.3.720-729.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schnaitman C. A. Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolate cell walls of Escherichia coli. J Bacteriol. 1971 Oct;108(1):553–563. doi: 10.1128/jb.108.1.553-563.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schnaitman C. A. Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels. Arch Biochem Biophys. 1973 Aug;157(2):541–552. doi: 10.1016/0003-9861(73)90673-5. [DOI] [PubMed] [Google Scholar]
  17. Showe M. K., DeMoss J. A. Localization and regulation of synthesis of nitrate reductase in Escherichia coli. J Bacteriol. 1968 Apr;95(4):1305–1313. doi: 10.1128/jb.95.4.1305-1313.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in Escherichia coli lacking cytochromes. Biochem J. 1975 May;148(2):329–333. [PMC free article] [PubMed] [Google Scholar]
  19. TANIGUCHI S., ITAGAKI E. Nitrate reductase of nitrate respiration type from E. coli. I. Solubilization and purification from the particulate system with molecular characterization as a metalloprotein. Biochim Biophys Acta. 1960 Nov 4;44:263–279. doi: 10.1016/0006-3002(60)91562-6. [DOI] [PubMed] [Google Scholar]
  20. Taylor C., Cox A. J., Kernohan J. C., Cohen P. Debranching enzyme from rabbit skeletal muscle. Purification, properties and physiological role. Eur J Biochem. 1975 Feb 3;51(1):105–115. doi: 10.1111/j.1432-1033.1975.tb03911.x. [DOI] [PubMed] [Google Scholar]
  21. Valentine R. C., Shapiro B. M., Stadtman E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry. 1968 Jun;7(6):2143–2152. doi: 10.1021/bi00846a017. [DOI] [PubMed] [Google Scholar]
  22. Van 't Riet J., Planta R. J. Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes. Biochim Biophys Acta. 1975 Jan 30;379(1):81–94. doi: 10.1016/0005-2795(75)90010-0. [DOI] [PubMed] [Google Scholar]
  23. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES