Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1974 Oct;144(1):77–85. doi: 10.1042/bj1440077

Energy-linked reduction of nicotinamide–adenine dinucleotide in membranes derived from normal and various respiratory-deficient mutant strains of Escherichia coli K12

Robert K Poole 1, Bruce A Haddock 1
PMCID: PMC1168466  PMID: 4156832

Abstract

1. Assay conditions are described for the ATP-dependent, uncoupler-sensitive, energy-linked reduction of NAD+ by succinate, dl-α-glycerophosphate or d-lactate in membranes from aerobically grown Escherichia coli. 2. The reaction may be demonstrated in electron-transport particles (ET particles) from cells grown in glycerol, but not in depleted particles washed in low-ionic-strength buffer, or in ET particles from cells grown in glucose. 3. The latter two classes of particles have low specific activities of ATPase (adenosine triphosphatase), succinate dehydrogenase, dl-α-glycerophosphate dehydrogenase and d-lactate dehydrogenase relative to undepleted ET particles from cells grown in glycerol. 4. Reconstitution of energy-linked NAD+ reduction in particles from cells grown in glucose was done by: (a) addition of the high-speed supernatant fraction from sonicates of the same cells; (b) addition of a protein fraction, precipitated by (NH4)2SO4 from this supernatant, or (c) addition of an (NH4)2SO4-precipitated fraction from the low-ionic-strength wash of particles from cells grown in glycerol. 5. The use of (NH4)2SO4-precipitated fractions from ATPase- or succinate dehydrogenase-deficient mutants grown in glycerol in the above reconstitution indicated that failure to demonstrate the reaction in particles from cells grown in glucose was a result of inadequate activities of appropriate dehydrogenases, rather than of ATPase. 6. Energy-linked NAD+ reduction could be demonstrated in particles from a ubiquinone-deficient mutant only after restoration of NADH oxidase activity by adding ubiquinone-1. 7. The measured rate of the energy-linked reaction in particles from a haem-deficient mutant, however, was not stimulated after the ATP- and haematin-dependent acquisition of functional cytochromes. 8. Results are interpreted as evidence of the ubiquinone-dependent, but cytochrome-independent, nature of the site I region of the respiratory chain in E. coli.

Full text

PDF
77

Selected References

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

  1. ARRIGONI O., SINGER T. P. Limitations of the phenazine methosulphate assay for succinic and related dehydrogenases. Nature. 1962 Mar 31;193:1256–1258. doi: 10.1038/1931256a0. [DOI] [PubMed] [Google Scholar]
  2. Altmann F. P. The use of eight different tetrazolium salts for a quantitative study of pentose shunt dehydrogenation. Histochemie. 1969;19(4):363–374. doi: 10.1007/BF00279685. [DOI] [PubMed] [Google Scholar]
  3. Asano A., Imai K., Sato R. Oxidative phosphorylation in Micrococcus denitrificans. 3. ATP-supported reduction of NAD+ by succinate. J Biochem. 1967 Aug;62(2):210–214. doi: 10.1093/oxfordjournals.jbchem.a128650. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bragg P. D., Hou C. Reconstitution of energy-dependent transhydrogenase in ATPase-negative mutants of Escherichia coli. Biochem Biophys Res Commun. 1973 Feb 5;50(3):729–736. doi: 10.1016/0006-291x(73)91305-3. [DOI] [PubMed] [Google Scholar]
  6. CHANCE B., HOLLUNGER G. Energy-linked reduction of mitochondrial pyridine nucleotide. Nature. 1960 Mar 5;185:666–672. doi: 10.1038/185666a0. [DOI] [PubMed] [Google Scholar]
  7. COLOWICK S. P., KAPLAN N. O., CIOTTI M. M. The reaction of pyridine nucleotide with cyanide and its analytical use. J Biol Chem. 1951 Aug;191(2):447–459. [PubMed] [Google Scholar]
  8. Cox G. B., Gibson F. Studies on electron transport and energy-linked reactions using mutants of Escherichia coli. Biochim Biophys Acta. 1974 Apr 30;346(1):1–25. doi: 10.1016/0304-4173(74)90010-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Cox G. B., Newton N. A., Gibson F., Snoswell A. M., Hamilton J. A. The function of ubiquinone in Escherichia coli. Biochem J. 1970 Apr;117(3):551–562. doi: 10.1042/bj1170551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cox G. B., Young I. G., McCann L. M., Gibson F. Biosynthesis of ubiquinone in Escherichia coli K-12: location of genes affecting the metabolism of 3-octaprenyl-4-hydroxybenzoic acid and 2-octaprenylphenol. J Bacteriol. 1969 Aug;99(2):450–458. doi: 10.1128/jb.99.2.450-458.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haddock B. A. The reconstitution of oxidase activity in membranes derived from a 5-aminolaevulinic acid-requiring mutant of Escherichia coli. Biochem J. 1973 Dec;136(4):877–884. doi: 10.1042/bj1360877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hane M. W., Wood T. H. Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J Bacteriol. 1969 Jul;99(1):238–241. doi: 10.1128/jb.99.1.238-241.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hendler R. W., Burgess A. H. Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. VI. Solubilization and characterization of the electron transport chain. J Cell Biol. 1972 Nov;55(2):266–281. doi: 10.1083/jcb.55.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. KASHKET E. R., BRODIE A. F. OXIDATIVE PHOSPHORYLATION IN FRACTIONATED BACTERIAL SYSTEMS. VIII. ROLE OF PARTICULATE AND SOLUBLE FRACTIONS FROM ESCHERICHIA COLI. Biochim Biophys Acta. 1963 Oct 8;78:52–65. doi: 10.1016/0006-3002(63)91608-1. [DOI] [PubMed] [Google Scholar]
  16. KASHKET E. R., BRODIE A. F. Oxidative phosphorylation in fractionated bacterial systems. X. Different roles for the natural quinones of Escherichia coli W in oxidative metabolism. J Biol Chem. 1963 Jul;238:2564–2570. [PubMed] [Google Scholar]
  17. Kung H. F., Henning U. Limiting availability of binding sites for dehydrogenases on the cell membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1972 Apr;69(4):925–929. doi: 10.1073/pnas.69.4.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LIN E. C., KOCH J. P., CHUSED T. M., JORGENSEN S. E. Utilization of L-alpha-glycerophosphate by Escherichia coli without hydrolysis. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2145–2150. doi: 10.1073/pnas.48.12.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lawford H. G., Haddock B. A. Respiration-driven proton translocation in Escherichia coli. Biochem J. 1973 Sep;136(1):217–220. doi: 10.1042/bj1360217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. MacGregor C. H., Schnaitman C. A. Reconstitution of nitrate reductase activity and formation of membrane particles from cytoplasmic extracts of chlorate-resistant mutants of Escherichia coli. J Bacteriol. 1973 Jun;114(3):1164–1176. doi: 10.1128/jb.114.3.1164-1176.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mitchell P. Hypothesis: cation-translocating adenosine triphosphatase models: how direct is the participation of adenosine triphosphate and its hydrolysis products in cation translocation? FEBS Lett. 1973 Jul 15;33(3):267–274. doi: 10.1016/0014-5793(73)80209-1. [DOI] [PubMed] [Google Scholar]
  23. Newton N. A., Cox G. B., Gibson F. The function of menaquinone (vitamin K 2 ) in Escherichia coli K-12. Biochim Biophys Acta. 1971 Jul 20;244(1):155–166. doi: 10.1016/0304-4165(71)90132-2. [DOI] [PubMed] [Google Scholar]
  24. Pirt S. J. A kinetic study of the mode of growth of surface colonies of bacteria and fungi. J Gen Microbiol. 1967 May;47(2):181–197. doi: 10.1099/00221287-47-2-181. [DOI] [PubMed] [Google Scholar]
  25. Racker E. Reconstitution of cytochrome oxidase vesicles and conferral of sensitivity to energy transfer inhibitors. J Membr Biol. 1972 Dec 29;10(3):221–235. doi: 10.1007/BF01867856. [DOI] [PubMed] [Google Scholar]
  26. Ragan C. I., Racker E. Partial resolution of the enzymes catalyzing oxidative phosphorylation. 28. The reconstitution of the first site of energy conservation. J Biol Chem. 1973 Apr 10;248(7):2563–2569. [PubMed] [Google Scholar]
  27. Reeves J. P., Hong J. S., Kaback H. R. Reconstitution of D-lactate-dependent transport in membrane vesicles from a D-lactate dehydrogenase mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1917–1921. doi: 10.1073/pnas.70.7.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Spencer M. E., Guest J. R. Proteins of the inner membrane of Escherichia coli: identification of succinate dehydrogenase by polyacrylamide gel electrophoresis with sdh amber mutants. J Bacteriol. 1974 Mar;117(3):947–953. doi: 10.1128/jb.117.3.947-953.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stekhoven F. M., Sani B. P., Sanadi D. R. Activation energies for the ATP-driven reversal of oxidative phosphorylation in submitochondrial particles. Biochim Biophys Acta. 1971 Jan 12;226(1):20–32. doi: 10.1016/0005-2728(71)90174-5. [DOI] [PubMed] [Google Scholar]
  32. Sweetman A. J., Griffiths D. E. Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide-adenine dinucleotide by succinate in Escherichia coli. Biochem J. 1971 Jan;121(1):117–124. doi: 10.1042/bj1210117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taylor A. L., Trotter C. D. Linkage map of Escherichia coli strain K-12. Bacteriol Rev. 1972 Dec;36(4):504–524. doi: 10.1128/br.36.4.504-524.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. West I. C., Mitchell P. The proton-translocating ATPase of Escherichia coli. FEBS Lett. 1974 Mar 15;40(1):1–4. doi: 10.1016/0014-5793(74)80880-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES