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. 1974 Jul;71(7):2720–2724. doi: 10.1073/pnas.71.7.2720

Purification and Properties of Mg2+-Ca2+ Adenosinetriphosphatase from Escherichia coli

Nathan Nelson *, Baruch I Kanner †, David L Gutnick †
PMCID: PMC388540  PMID: 4277624

Abstract

A procedure for the purification of Mg2+-Ca2+ adenosinetriphosphatase (EC 3.6.1.3) from E. coli, yielding relatively large amounts of highly active enzyme, is described. The enzyme consists of four nonidentical subunits. Trypsin treatment of purified enzyme yields a preparation consisting exclusively of the two larger subunits, which are sufficient for ATPase activity. Purified enzyme is inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; this inhibition is reversed by dithiothreitol, and the diazole is found preferentially associated with the β-subunit of the enzyme. Antibody prepared against the trypsin-treated enzyme inhibited various ATP-dependent reactions as well as membrane-bound ATPase itself.

Keywords: sulfhydryl reagent, catalytic subunits, antibody

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Selected References

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

  1. Bragg P. D., Hou C. Purification of a factor for both aerobic-driven and ATP-driven energy-dependent transhydrogenases of Escherichia coli. FEBS Lett. 1972 Dec 15;28(3):309–312. doi: 10.1016/0014-5793(72)80738-5. [DOI] [PubMed] [Google Scholar]
  2. Butlin J. D., Cox G. B., Gibson F. Oxidative phosphorylation in Escherichia coli K-12: the genetic and biochemical characterisations of a strain carrying a mutation in the uncB gene. Biochim Biophys Acta. 1973 Feb 22;292(2):366–375. doi: 10.1016/0005-2728(73)90043-1. [DOI] [PubMed] [Google Scholar]
  3. Butlin J. D., Cox G. B., Gibson F. Oxidative phosphorylation in Escherichia coli K12. Mutations affecting magnesium ion- or calcium ion-stimulated adenosine triphosphatase. Biochem J. 1971 Aug;124(1):75–81. doi: 10.1042/bj1240075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cox G. B., Newton N. A., Butlin J. D., Gibson F. The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K12. Biochem J. 1971 Nov;125(2):489–493. doi: 10.1042/bj1250489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fisher R. J., Sanadi D. R. Energy-linked nicotinamide adenine dinucleotide transhydrogenase in membrane particles from Escherchia coli. Biochim Biophys Acta. 1971 Aug 6;245(1):34–41. doi: 10.1016/0005-2728(71)90005-3. [DOI] [PubMed] [Google Scholar]
  6. Giordana G., Riviere C., Azoulay E. Membrane reconstitution in chl-r mutants of Escherichia coli K 12. V. ATPase incorporation into particles formed by complementation. Biochim Biophys Acta. 1973 May 25;307(3):513–524. doi: 10.1016/0005-2736(73)90297-6. [DOI] [PubMed] [Google Scholar]
  7. Gutnick D. L., Kanner B. I., Postma P. W. Oxidative phosphorylation in mutants of Escherichia coli defective in energy transduction. Biochim Biophys Acta. 1972 Nov 17;283(2):217–222. doi: 10.1016/0005-2728(72)90237-x. [DOI] [PubMed] [Google Scholar]
  8. Hanson R. L., Kennedy E. P. Energy-transducing adenosine triphosphatase from Escherichia coli: purification, properties, and inhibition by antibody. J Bacteriol. 1973 May;114(2):772–781. doi: 10.1128/jb.114.2.772-781.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kanner B. I., Gutnick D. L. Energy linked nicotinamide adenine dinucleotide transhydrogenase in a mutant of Escherichia coli K12 lacking membrane Mg(2+)&z.sbnd;Ca(2+)-activated adenosine triphosphatase. FEBS Lett. 1972 May 1;22(2):197–199. doi: 10.1016/0014-5793(72)80043-7. [DOI] [PubMed] [Google Scholar]
  10. Kanner B. I., Gutnick D. L. Use of neomycin in the isolation of mutants blocked in energy conservation in Escherichia coli. J Bacteriol. 1972 Jul;111(1):287–289. doi: 10.1128/jb.111.1.287-289.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kobayashi H., Anraku Y. Membrane-bound adenosine triphosphatase of Escherichia coli. I. Partial purification and properties. J Biochem. 1972 Mar;71(3):387–399. [PubMed] [Google Scholar]
  12. 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]
  13. Nelson N., Deters D. W., Nelson H., Racker E. Partial resolution of the enzymes catalyzing photophosphorylation. 8. Properties of isolated subunits of coupling factor 1 from spinach chloroplasts. J Biol Chem. 1973 Mar 25;248(6):2049–2055. [PubMed] [Google Scholar]
  14. Nelson N., Nelson H., Racker E. Partial resolution of the enzymes catalyzing photophosphorylation. XI. Magnesium-adenosine triphosphatase properties of heat-activated coupling factor I from chloroplasts. J Biol Chem. 1972 Oct 25;247(20):6506–6510. [PubMed] [Google Scholar]
  15. Nieuwenhuis F. J., Kanner B. I., Gutnick D. L., Postma P. W., van Dam K. Energy conservation in membranes of mutants of Escherichia coli defective in oxidative phosphorylation. Biochim Biophys Acta. 1973 Oct 19;325(1):62–71. doi: 10.1016/0005-2728(73)90151-5. [DOI] [PubMed] [Google Scholar]
  16. Roisin M. P., Kepes A. The membrane ATPase of Escherichia coli. I. Release into solution, allotopic properties and reconstitution of membrane-bound ATPase. Biochim Biophys Acta. 1973 May 30;305(2):249–259. doi: 10.1016/0005-2728(73)90173-4. [DOI] [PubMed] [Google Scholar]
  17. Schairer H. U., Gruber D. Mutants of Escherichia coli K12 defective in oxidative phosphorylation. Eur J Biochem. 1973 Aug 17;37(2):282–286. doi: 10.1111/j.1432-1033.1973.tb02986.x. [DOI] [PubMed] [Google Scholar]
  18. Schairer H. U., Haddock B. A. -Galactoside accumulation in a Mg 2+ -,Ca 2+ -activated ATPase deficient mutant of E.coli. Biochem Biophys Res Commun. 1972 Aug 7;48(3):544–551. doi: 10.1016/0006-291x(72)90382-8. [DOI] [PubMed] [Google Scholar]
  19. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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