Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1983 Feb 15;210(2):395–403. doi: 10.1042/bj2100395

Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

A E Senior, L Langman, G B Cox, F Gibson
PMCID: PMC1154237  PMID: 6222731

Abstract

To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

Full text

PDF
395

Selected References

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

  1. Cox G. B., Downie J. A., Gibson F., Radik J. Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. Biochem J. 1978 Mar 15;170(3):593–598. doi: 10.1042/bj1700593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cox G. B., Downie J. A. Isolation and characterization of mutants of Escherichia coli K-12 affected in oxidative phosphorylation of quinone biosynthesis. Methods Enzymol. 1979;56:106–117. doi: 10.1016/0076-6879(79)56013-3. [DOI] [PubMed] [Google Scholar]
  3. Cox G. B., Downie J. A., Langman L., Senior A. E., Ash G., Fayle D. R., Gibson F. Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits. J Bacteriol. 1981 Oct;148(1):30–42. doi: 10.1128/jb.148.1.30-42.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cox G. B., Gibson F., McCann L. Reconstitution of oxidative phosphorylation and the adenosine triphosphate-dependent transhydrogenase activity by a combination of membrane fractions from unCA- and uncB- mutant strains of Escherichia coli K12. Biochem J. 1973 Aug;134(4):1015–1021. doi: 10.1042/bj1341015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cross R. L. The mechanism and regulation of ATP synthesis by F1-ATPases. Annu Rev Biochem. 1981;50:681–714. doi: 10.1146/annurev.bi.50.070181.003341. [DOI] [PubMed] [Google Scholar]
  6. Downie J. A., Gibson F., Cox G. B. Membrane adenosine triphosphatases of prokaryotic cells. Annu Rev Biochem. 1979;48:103–131. doi: 10.1146/annurev.bi.48.070179.000535. [DOI] [PubMed] [Google Scholar]
  7. Downie J. A., Langman L., Cox G. B., Yanofsky C., Gibson F. Subunits of the adenosine triphosphatase complex translated in vitro from the Escherichia coli unc operon. J Bacteriol. 1980 Jul;143(1):8–17. doi: 10.1128/jb.143.1.8-17.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Esch F. S., Allison W. S. Identification of a tyrosine residue at a nucleotide binding site in the beta subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosine. J Biol Chem. 1978 Sep 10;253(17):6100–6106. [PubMed] [Google Scholar]
  9. Esch F. S., Böhlen P., Otsuka A. S., Yoshida M., Allison W. S. Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit. J Biol Chem. 1981 Sep 10;256(17):9084–9089. [PubMed] [Google Scholar]
  10. Fayle D. R., Downie J. A., Cox G. B., Gibson F., Radik J. Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered beta-subunit of the magnesium ion-stimulated adenosine triphosphatase. Biochem J. 1978 Jun 15;172(3):523–531. doi: 10.1042/bj1720523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson F., Cox G. B., Downie J. A., Radik J. A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele. Biochem J. 1977 Apr 15;164(1):193–198. doi: 10.1042/bj1640193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gibson F., Downie J. A., Cox G. B., Radik J. Mu-induced polarity in the unc operon of Escherichia coli. J Bacteriol. 1978 Jun;134(3):728–736. doi: 10.1128/jb.134.3.728-736.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grubmeyer C., Penefsky H. S. The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1981 Apr 25;256(8):3718–3727. [PubMed] [Google Scholar]
  14. Gunsalus R. P., Brusilow W. S., Simoni R. D. Gene order and gene-polypeptide relationships of the proton-translocating ATPase operon (unc) of Escherichia coli. Proc Natl Acad Sci U S A. 1982 Jan;79(2):320–324. doi: 10.1073/pnas.79.2.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kanazawa H., Horiuchi Y., Takagi M., Ishino Y., Futai M. Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli. J Biochem. 1980 Sep;88(3):695–703. doi: 10.1093/oxfordjournals.jbchem.a133022. [DOI] [PubMed] [Google Scholar]
  16. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  17. Senior A. E., Downie J. A., Cox G. B., Gibson F., Langman L., Fayle D. R. The uncA gene codes for the alpha-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains. Biochem J. 1979 Apr 15;180(1):103–109. doi: 10.1042/bj1800103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Senior A. E., Fayle D. R., Downie J. A., Gibson F., Cox G. B. Properties of membranes from mutant strains of Escherichia coli in which the beta-subunit of the adenosine triphosphatase is abnormal. Biochem J. 1979 Apr 15;180(1):111–118. doi: 10.1042/bj1800111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Senior A. E. Tightly bound magnesium in mitochondrial adenosine triphosphatase from beef heart. J Biol Chem. 1979 Nov 25;254(22):11319–11322. [PubMed] [Google Scholar]
  20. Wise J. G., Latchney L. R., Senior A. E. The defective proton-ATPase of uncA mutants of Escherichia coli. Studies of nucleotide binding sites, bound aurovertin fluorescence, and labeling of essential residues of the purified F1-ATPase. J Biol Chem. 1981 Oct 25;256(20):10383–10389. [PubMed] [Google Scholar]

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

RESOURCES