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J Bacteriol. Feb 1979; 137(2): 719–726.
PMCID: PMC218348

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

Abstract

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [99Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated 99Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, 99Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of 99Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [99Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.

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Selected References

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  • DAVIS BJ. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. [PubMed]
  • Enoch HG, Lester RL. The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli. J Biol Chem. 1975 Sep 10;250(17):6693–6705. [PubMed]
  • Glaser JH, DeMoss JA. Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli. J Bacteriol. 1971 Nov;108(2):854–860. [PMC free article] [PubMed]
  • Jones HP, Johnson JL, Rajagopalan KV. In vitro reconstitution of demolybdosulfite oxidase by molybdate. J Biol Chem. 1977 Jul 25;252(14):4988–4993. [PubMed]
  • Ketchum PA, Cambier HY, Frazier WA, 3rd, Madansky CH, 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. [PMC free article] [PubMed]
  • LENNOX ES. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. [PubMed]
  • Lester RL, DeMoss JA. Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli. J Bacteriol. 1971 Mar;105(3):1006–1014. [PMC free article] [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Lund K, DeMoss JA. Association-dissociation behavior and subunit structure of heat-released nitrate reductase from Escherichia coli. J Biol Chem. 1976 Apr 25;251(8):2207–2216. [PubMed]
  • Nagatani HH, Shah VK, Brill WJ. Activation of inactive nitrogenase by acid-treated component I. J Bacteriol. 1974 Nov;120(2):697–701. [PMC free article] [PubMed]
  • Nason A, Antoine AD, Ketchum PA, Frazier WA, 3rd, Lee DK. Formation of assimilatory nitrate reductase by in vitro inter-cistronic complementation in Neurospora crassa. Proc Natl Acad Sci U S A. 1970 Jan;65(1):137–144. [PMC free article] [PubMed]
  • Nason A, Lee KY, Pan SS, Ketchum PA, 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. [PMC free article] [PubMed]
  • Pienkos PT, Shah VK, Brill WJ. Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5468–5471. [PMC free article] [PubMed]
  • Ruiz-Herrera J, DeMoss JA. 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. [PMC free article] [PubMed]
  • Ruiz-Herrera J, Showe MK, DeMoss JA. Nitrate reductase complex of Escherichia coli K-12: isolation and characterization of mutants unable to reduce nitrate. J Bacteriol. 1969 Mar;97(3):1291–1297. [PMC free article] [PubMed]
  • Scott RH, DeMoss JA. Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli. J Bacteriol. 1976 Apr;126(1):478–486. [PMC free article] [PubMed]
  • Showe MK, DeMoss JA. Localization and regulation of synthesis of nitrate reductase in Escherichia coli. J Bacteriol. 1968 Apr;95(4):1305–1313. [PMC free article] [PubMed]
  • Sperl GT, DeMoss JA. chlD gene function in molybdate activation of nitrate reductase. J Bacteriol. 1975 Jun;122(3):1230–1238. [PMC free article] [PubMed]
  • 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. [PubMed]

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