scholarly journals Oxidative phosphorylation by mutant Escherichia coli membranes with impaired proton permeability

1983 ◽  
Vol 216 (1) ◽  
pp. 143-150 ◽  
Author(s):  
G B Cox ◽  
D A Jans ◽  
F Gibson ◽  
L Langman ◽  
A E Senior ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Gene Dosage ◽  
Control Strain ◽  
Proton Permeability ◽  
F1f0 Atpase ◽  
F1 Atpase ◽  
C Subunit

The effect on the function of the Escherichia coli F1F0-ATPase of the substitution of leucine-31 by phenylalanine in the c-subunit of the enzyme was examined. The assembly of the mutant c-subunit requires an increased gene dosage [Jans, Fimmel, Langman, James, Downie, Senior, Ash, Gibson & Cox (1983) Biochem. J. 211, 717-726], and this was achieved by incorporation of the uncE408 or uncE463 alleles on to F-plasmids or multicopy plasmids. Membranes from strains carrying either the uncE463 or uncE408 alleles on F-plasmids or multicopy plasmids were capable of carrying out oxidative phosphorylation. In particular, membranes from strain AN1928 (pAN162, uncE463) gave phosphorylation rates and P/O ratios equal to or greater than those obtained for the control strain AN1460 (pAN45, unc+). However, the mutant membranes, on removal of the F1-ATPase, appeared to be proton-impermeable. The ATPase activity of the mutant membranes was also resistant to the inhibitor dicyclohexylcarbodi-imide.

scholarly journals The F1F0-ATPase of Escherichia coli. Substitution of proline by leucine at position 64 in the c-subunit causes loss of oxidative phosphorylation

1983 ◽  
Vol 213 (2) ◽  
pp. 451-458 ◽  
Author(s):  
A L Fimmel ◽  
D A Jans ◽  
L Langman ◽  
L B James ◽  
G R Ash ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Base Change ◽  
Helix Axis ◽  
F1f0 Atpase ◽  
F1 Atpase ◽  
C Subunit

The uncE410 allele differs from the normal uncE gene in that C leads to T base changes occur at nucleotides 190 and 191, resulting in proline at position 64 in the c-subunit of the F1F0-ATPase being replaced by leucine. Two partial-revertant strains were isolated in which alanine-20 of the c-subunit was replaced by proline, owing to a G leads to C base change at nucleotide 58. These c-subunits, coded for by the uncE501 and uncE502 alleles, therefore contained two amino acid changes, namely proline-64 leads to leucine, and alanine-20 leads to proline. Membranes prepared from the partial-revertant strains lacked ATP-dependent atebrin-fluorescence-quenching activity but were able to carry out oxidative phosphorylation. The ATPase activity of the F1-ATPase was inhibited when bound to membranes from strains carrying the uncE410, uncE501 and uncE502 alleles. It is concluded that a bend in the helix axis in one of the arms of the c-subunit hairpin structure is required for integration of the c-subunit into a functional F1F0-ATPase.

scholarly journals Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation

1977 ◽  
Vol 162 (3) ◽  
pp. 665-670 ◽  
Author(s):  
F Gibson ◽  
G B Cox ◽  
J A Downie ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Growth Yields ◽  
Normal Allele ◽  
Adenosine Triphosphatase Activity

A plasmid was isolated which included the region of the Escherichia coli chromosome carrying the known genes concerned with oxidative phosphorylation (unc genes). This plasmid was used to prepare partial diploids carrying normal unc alleles on the episome and one of the three mutant alleles (unc A401, uncB402 or unc-405) on the chromosome. These strains were compared with segregants from which the plasmid had been lost. Dominance of either normal ormutant unc alleles was determined by growth on succinate, growth yields on glucose, Mg-ATPase (Mg2+-stimulated adenosine triphosphatase) activity, atebrin-fluorescence quenching, ATP-dependent transhydrogenase activity and oxidative phosphorylation. In all the above tests, dominance of the normal allele was observed. However, in membranes from the diploid strains which carried a normal allele and either of the mutant alleles affecting Mg-ATPase activity (uncA401 or unc-405), the energy-linked functions were only partially restored.

scholarly journals Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli

1983 ◽  
Vol 211 (3) ◽  
pp. 717-726 ◽  
Author(s):  
D A Jans ◽  
A L Fimmel ◽  
L Langman ◽  
L B James ◽  
J A Downie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cell Membrane ◽  
Dna Sequences ◽  
Adenosine Triphosphatase ◽  
Gene Dosage ◽  
Base Change ◽  
Amino Acid Substitutions ◽  
Helical Hairpin ◽  
C Subunit

The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the ‘Helical Hairpin Hypothesis’ of Engelman & Steitz [(1981) Cell 23,411-422].

The F1F0-ATPase of Escherichia coli. The substitution of alanine by threonine at position 25 in the c-subunit affects function but not assembly

1985 ◽  
Vol 808 (2) ◽  
pp. 252-258 ◽  
Author(s):  
Anthony L. Fimmel ◽  
David A. Jans ◽  
Lyndall Hatch ◽  
Lewis B. James ◽  
Frank Gibson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
F1f0 Atpase ◽  
C Subunit

Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase

Biochemistry ◽  
1990 ◽  
Vol 29 (45) ◽  
pp. 10387-10393 ◽  
Author(s):  
Robert Aggeler ◽  
Janet Mendel-Hartvig ◽  
Roderick A. Capaldi
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Atpase Activity ◽  
F1 Atpase

Enhancement of glucose metabolism in a pyruvic acid-hyperproducing Escherichia coli mutant defective in F1-ATPase activity

1997 ◽  
Vol 83 (2) ◽  
pp. 132-138 ◽  
Author(s):  
Atsushi Yokota ◽  
Masaru Henmi ◽  
Naohisa Takaoka ◽  
Chiho Hayashi ◽  
Yuji Takezawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glucose Metabolism ◽  
Atpase Activity ◽  
Pyruvic Acid ◽  
F1 Atpase ◽  
Coli Mutant

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

1983 ◽  
Vol 210 (2) ◽  
pp. 395-403 ◽  
Author(s):  
A E Senior ◽  
L Langman ◽  
G B Cox ◽  
F Gibson
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Atpase Activity ◽  
Atp Synthesis ◽  
Beta Subunit ◽  
F1 Atpase ◽  
Dependent Atpase ◽  
Mutant Strains ◽  
Synthesis Activity ◽  
Low Ionic Strength

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.

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