scholarly journals Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the F1 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12

1978 ◽  
Vol 170 (3) ◽  
pp. 593-598 ◽  
Author(s):  
G B Cox ◽  
J A Downie ◽  
F Gibson ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
The Other ◽  
Genetic Complementation ◽  
Magnesium Ion ◽  
Adenosine Triphosphatase Activity

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.

scholarly journals A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele

1977 ◽  
Vol 164 (1) ◽  
pp. 193-198 ◽  
Author(s):  
F Gibson ◽  
G B Cox ◽  
J A Downie ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Fluorescence Quenching ◽  
Mutant Strain ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Magnesium Ion ◽  
New Mutation ◽  
Similar Phenotype

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.

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 Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1973 ◽  
Vol 132 (4) ◽  
pp. 689-695 ◽  
Author(s):  
G. B. Cox ◽  
F. Gibson ◽  
L. M. McCann ◽  
J. D. Butlin ◽  
F. L. Crane
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Calcium Ion ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Gel Filtration ◽  
Magnesium Ion ◽  
Adenosine Triphosphatase Activity ◽  
Escherichia Coli K12

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg2+,Ca2+-stimulated adenosine triphosphatase. 2. The Mg2+,Ca2+-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg2+,Ca2+-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg2+,Ca2+-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg2+,Ca2+-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA-) could not be re-activated by the purified Mg2+,Ca2+-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg2+,Ca2+-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA-).

scholarly journals An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function

1984 ◽  
Vol 221 (1) ◽  
pp. 43-51 ◽  
Author(s):  
D A Jans ◽  
A L Fimmel ◽  
L Hatch ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Dna Sequence ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
The Other ◽  
Multicopy Plasmid ◽  
B Subunit ◽  
And Function

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.

scholarly journals Oxidative phosphorylation in Escherichia coli K12. An uncoupled mutant with altered membrane structure

1974 ◽  
Vol 138 (2) ◽  
pp. 211-215 ◽  
Author(s):  
G. B. Cox ◽  
F. Gibson ◽  
L. McCann
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Membrane Structure ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Mineral Salts ◽  
Normal Cells ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Membrane Bound

1. A new mutant strain (AN228) of Escherichia coli K12, unable to couple phosphorylation to electron transport, has been isolated. The mutant allele (unc-405), in strain AN228, was found to map near the uncA and uncB genes at about minute 74 on the E. coli genome. 2. A transductant strain (AN285) carrying the unc-405 allele is similar to the uncA and uncB mutants described previously in that it is unable to grow on succinate, gives a low aerobic yield on limiting concentrations of glucose, has a normal rate of electron transport, is unable to couple phosphorylation to electron transport, and lacks ATP-dependent transhydrogenase activity. 3. Strain AN285 (unc-405) is similar to an uncA mutant, but different from an uncB mutant, in that it is unable to grow anaerobically in a glucose–mineral-salts medium, and membrane preparations do not have Mg2+-stimulated adenosine triphosphatase activity. 4. Strain AN285 (unc-405) does not form an aggregate analogous to the membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate found in normal cells. In this respect it differs from strain AN249 (uncA−), which forms an inactive membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate.

scholarly journals ADENOSINE TRIPHOSPHATASE: A NEUROHISTOCHEMICAL STUDY

1962 ◽  
Vol 10 (6) ◽  
pp. 731-740 ◽  
Author(s):  
D. NAIDOO
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Wistar Rat ◽  
The Other ◽  
Nuclear Staining ◽  
Adenosine Triphosphatase Activity ◽  
Bivalent Cations ◽  
Vascular Elements ◽  
The Brain ◽  
Cerebral Vascular

The location of adenosine triphosphatase in the brain has been studied in rapidly frozen-dried cerebral tissues of the Wistar rat. It is found that adenosine triphosphatase is an almost exclusively nuclear enzyme. Two tissue fractions of the cerebrum were separated, so that one sample was made up of vascular elements, and the other of neural elements. The two fractions were then studied for their adenosine triphosphatase activity, and compared with the histochemical findings. The two tissue fractions were found not to differ in the absence of bivalent cations. When Ca++ were added to the cerebral vascular suspension, ATPase activity was increased approximately 15 times, and only 3 times in the presence of Mg++. Conversely, the addition of Mg++ increased the ATPase activity of the neural fraction 200%; whereas, Ca++ was responsible for a 60% increase. This fact was detectable microscopically when Ca++ was found to intensify vascular nuclear staining, and Mg++ to increase the neuronal and glial nuclear staining. The results, histochemical and biochemical, are mutually confirmatory.

scholarly journals Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions

1966 ◽  
Vol 100 (3) ◽  
pp. 762-767 ◽  
Author(s):  
N Gruener ◽  
Y Avi-Dor
Keyword(s):  
Rat Brain ◽  
Adenosine Triphosphatase ◽  
Arrhenius Plot ◽  
Enzyme System ◽  
The Other ◽  
Potassium Ions ◽  
Adenosine Triphosphatase Activity ◽  
Brain Microsomes ◽  
High Concentrations ◽  
Sodium And Potassium

1. The adenosine-triphosphatase activity of rat-brain microsomes was measured between 0 degrees and 37 degrees . The stimulatory effect of Na(+) plus K(+) on the Mg(2+)-dependent adenosine-triphosphatase activity decreased sharply with decreasing temperature and became negligible at 0 degrees . An Arrhenius plot drawn from the experimental data showed two discontinuities: one at about 6 degrees and the other at about 20 degrees . 2. The increment in activity induced by Na(+) plus K(+) was more sensitive to oligomycin at lower than at higher temperatures, but the opposite was observed for ouabain. The action of oligomycin showed a biphasic character, since below a certain concentration it caused slight activation of Na(+)-plus-K(+)-activated adenosine triphosphatase. 3. Where oligomycin increased the activity of the enzyme, it also enhanced the accumulation of an acid-precipitable phosphorylated compound formed through the transfer of the gamma-phosphate group of [(32)P]ATP to the enzyme system. Stimulatory concentrations of oligomycin did not interfere with K(+)-mediated dephosphorylation of the intermediate, though high concentrations of oligomycin counteracted the effect of K(+). 4. The temperature profile of K(+)-stimulated microsomal phosphatase qualitatively resembled that of microsomal adenosine triphosphatase.

Localization of adenosine triphosphatase activity in motile bacteria

1973 ◽  
Vol 19 (10) ◽  
pp. 1265-1267 ◽  
Author(s):  
Z. Vaituzis
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Uniform Distribution ◽  
Adenosine Triphosphatase ◽  
Periplasmic Space ◽  
Reaction Products ◽  
Adenosine Triphosphatase Activity ◽  
Atpase Reaction ◽  
Motile Bacteria

Cytochemical studies on motile bacteria revealed magnesium-dependent adenosine triphosphatase (ATPase) activity at the membranous sites of flagellar origin. The studies were done on bacteria representing three types of flagellation, namely, peritrichate, lophotrichate, and monotrichate. Escherichia coli and S. serpens showed a uniform distribution of ATPase reaction products throughout the periplasmic space. In B. licheniformis and V. metchnikovii the reaction products were found in the cytoplasm accumulated in areas where flagella originate.

scholarly journals HISTOCHEMICAL DEMONSTRATION OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE ACTIVITY IN TISSUE SECTIONS

1962 ◽  
Vol 10 (1) ◽  
pp. 65-74 ◽  
Author(s):  
MAX WACHSTEIN ◽  
MAIRE BRADSHAW ◽  
JOSÉ M. ORTIZ
Keyword(s):  
Adenosine Triphosphatase ◽  
Histochemical Demonstration ◽  
The Other ◽  
Mitochondrial Activity ◽  
Bile Canaliculi ◽  
Adenosine Triphosphatase Activity ◽  
Tissue Sections ◽  
Dog Kidney ◽  
Cryostat Sections ◽  
Neutral Formalin

A comparative study was made of the distribution of mitochondrial adenosine triphosphatase activity in several organs of rat and rabbit using both the calcium and lead techniques. Certain modifications in these techniques assured both improved intracellular morphology and considerable preservation of enzyme activity. Of particular importance was the low temperature (–2 to –3°C) of the neutral calcium-formol solution, and, following short fixation (lead method), a subsequent wash in neutral buffer. Mitochondrial activity was similar with both techniques, but the lead method proved to be less costly, less time-consuming, and above all, far less capricious than the calcium technique. In good preparations, the appearance of mitochondria is as clearly defined as in sections stained with non-enzymatic, conventional techniques. Long exposure of cryostat sections to formalin or preparation of sections from tissue blocks fixed in neutral formalin leads to the complete abolition of mitochondrial activity; on the other hand, it accentuates enzyme staining of other structures, such as, for instance, bile canaliculi in the liver and the secretory capillaries in the pancreas and salivary glands. It also visualizes the infolding membranes in certain tubules of the rat and dog kidney. It is assumed that formalin-fixation aids in the enzymato-morphologic distinction of these two different intracellular structures.

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