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. 1973 Jun;114(3):1164–1176. doi: 10.1128/jb.114.3.1164-1176.1973

Reconstitution of Nitrate Reductase Activity and Formation of Membrane Particles from Cytoplasmic Extracts of Chlorate-Resistant Mutants of Escherichia coli

C H MacGregor 1, C A Schnaitman 1
PMCID: PMC285378  PMID: 4576401

Abstract

The reconstitution of nitrate reductase activity in mixtures of cytoplasmic fractions from the chlorate-resistant mutants chlA, B, C, and E which are lacking this activity was investigated, and the membrane-like particulate material which formed during this reconstitution was analyzed by polyacrylamide gel electrophoresis. When chlA and chlB extracts are incubated together, the cytoplasmic membrane proteins present in the particles which are formed are contributed by both mutants, and the proteins are essentially the same as the proteins in the cytoplasmic membrane fractions of the two mutants. Identical amounts of protein become particulate when cytoplasmic extracts of any of the mutant strains or wild-type strains are incubated at 32 C either singly or in mixtures, and the formation of particulate material does not appear to be a consequence of nitrate reductase reconstitution. Experiments with wild-type strains indicate that the membrane proteins in the cytoplasmic extract are derived from the cytoplasmic membrane during cell breakage. Reconstitution experiments involving various combinations of preincubated and unincubated extracts of the mutants have allowed a preliminary identification of three types of components which are necessary for the formation of active nitrate reductase: (i) a soluble factor present only in extracts from induced chlB; (ii) a different soluble factor which is missing in chlB but is present in extracts from wild-type, chlA, chlC, and chlE; and (iii) a complex including the nitrate reductase protein which is inactivated by preincubation of the mutant extracts.

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Selected References

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

  1. Azoulay E., Puig J., Couchoud-Beaumont P. Etude des mutants chlorate-résistants chez Escherichia coli K 12. I. Reconstitution in vitro de l'activité nitrate-réductase particulaire chez Escherichia coli K 12. Biochim Biophys Acta. 1969 Feb 11;171(2):238–252. doi: 10.1016/0005-2744(69)90157-0. [DOI] [PubMed] [Google Scholar]
  2. Azoulay E., Puig J., Pichinoty F. Alteration of respiratory particles by mutation in Escherichia coli K 12. Biochem Biophys Res Commun. 1967 Apr 20;27(2):270–274. doi: 10.1016/s0006-291x(67)80073-1. [DOI] [PubMed] [Google Scholar]
  3. Glaser J. H., DeMoss J. A. Comparison of nitrate reductase mutants of Escherichia coli selected by alternative procedures. Mol Gen Genet. 1972;116(1):1–10. doi: 10.1007/BF00334254. [DOI] [PubMed] [Google Scholar]
  4. Glaser J. H., DeMoss J. A. Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli. J Bacteriol. 1971 Nov;108(2):854–860. doi: 10.1128/jb.108.2.854-860.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Guest J. R. Biochemical and genetic studies with nitrate reductase C-gene mutants of Escherichia coli. Mol Gen Genet. 1969;105(4):285–297. doi: 10.1007/BF00277583. [DOI] [PubMed] [Google Scholar]
  6. Ketchum P. A., Cambier H. Y., Frazier W. A., 3rd, Madansky C. H., Nason A. In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals. Proc Natl Acad Sci U S A. 1970 Jul;66(3):1016–1023. doi: 10.1073/pnas.66.3.1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Lester R. L., DeMoss J. A. Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli. J Bacteriol. 1971 Mar;105(3):1006–1014. doi: 10.1128/jb.105.3.1006-1014.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. MacGregor C. H., Schnaitman C. A. Restoration of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase activity of a Neurospora mutant by extracts of various chlorate-resistant mutants of Escherichia coli. J Bacteriol. 1972 Oct;112(1):388–391. doi: 10.1128/jb.112.1.388-391.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nason A., Lee K. Y., Pan S. S., Ketchum P. A., Lamberti A., DeVries J. Invitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3242–3246. doi: 10.1073/pnas.68.12.3242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Riviere C., Azoulay E. Reconstitution in vitro of membranous particles by complementation between extracts of chl-r mutants in Escherichia coli K12. Biochem Biophys Res Commun. 1971 Dec 17;45(6):1608–1614. doi: 10.1016/0006-291x(71)90205-1. [DOI] [PubMed] [Google Scholar]
  13. Schnaitman C. A. Alteration of membrane proteins in a chlorate-resistant mutant of Escherichia coli. Biochem Biophys Res Commun. 1969 Sep 24;37(1):1–5. doi: 10.1016/0006-291x(69)90871-7. [DOI] [PubMed] [Google Scholar]
  14. Schnaitman C. A. Examination of the protein composition of the cell envelope of Escherichia coli by polyacrylamide gel electrophoresis. J Bacteriol. 1970 Nov;104(2):882–889. doi: 10.1128/jb.104.2.882-889.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]
  18. 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]

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