[16] Phosphofruktokinase from Escherichia coli

Kemerer, Verne F.; Griffin, Charles C.; Brand, Ludwig

doi:10.1016/0076-6879(75)42099-7

Cited by 15 publications

(7 citation statements)

References 9 publications

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“…Phosphofructokinase (EC 2.7.1.11) was measured as NADH decrease coupled to aldolase, triosephosphate isomerase and glycerophosphate dehydrogenase (modified after Kemerer et al, 1975). The assay consisted of 75 mM Tris-HCl, pH 7.6, 0.43 mM NADH, 1.3 mM MgCl2, 1.7 mM ATP, 0.7 U ml -1 aldolase (rabbit muscle), 25 U ml -1 triosephosphate isomerase, 2.5 U ml -1 glycerophosphate dehydrogenase (rabbit muscle) and 10-50 ml of cell-free extract.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Müller

Griffin

Stingl

et al. 2008

Environmental Microbiology

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Six strains of novel bacteria were isolated from profundal sediment of Lake Constance, a deep freshwater lake in Germany, by direct dilution of the sediment in mineral agar medium containing a background lawn of the hydrogen-scavenging Methanospirillum hungatei as a syntrophic partner. The numbers of colony-forming units obtained after incubation for more than 2 months were in the same range as those of total bacterial counts determined by DAPI staining (up to 10(8) cells per millilitre) suggesting that these organisms were dominant members of the community. Identical dilution series in the absence of methanogenic partners yielded numbers that were lower by two to three orders of magnitude. The dominant bacteria were isolated in defined co-culture with M. hungatei, and were further characterized. Growth was slow, with doubling times of 22-28 h at 28 degrees C. Cells were small, 0.5 x 5 microm in size, Gram-positive, and formed terminal oval spores. At 20 degrees C, glucose was fermented by the co-culture strain BoGlc83 nearly stoichiometrically to 2 mol of acetate and 1 mol of methane plus CO(2). At higher temperatures, also lactate and traces of succinate were formed. Anaerobic growth depended strictly on the presence of a hydrogen-scavenging partner organism and was inhibited by bromoethane sulfonate, which together indicate the need for a syntrophic partnership for this process. Strain BoGlc83 grew also aerobically in the absence of a partner organism. All enzymes involved in ATP formation via glycolysis and acetyl CoA were found, most of them at activities equivalent to the physiological substrate turnover rate. This new type of sugar-fermenting bacterium appears be the predominant sugar utilizer in this environment. The results show that syntrophic relationships can play an important role also for the utilization of substrates which otherwise can be degraded in pure culture.

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Section: Enzyme Assaysmentioning

confidence: 99%

Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Müller

Griffin

Stingl

et al. 2008

Environmental Microbiology

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“…These conclusions are unlikely to be materially altered if PEP is decarboxylated by an alternative enzyme, such as PEP carboxylase [orthophosphate-oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31]. In addition, pyruvate lies outside the major feedback loop in both the control [8] and PFK transgenic potato lines [16,17], namely that of PEP inhibition of PFK, and is therefore likely to respond in a different manner from the other glycolytic intermediates (see the Discussion). Finally, there is the additional partitioning of pyruvate into the mitochondria to consider, although this is likely to be a relatively minor effect as mitochondrial pyruvate is probably a small proportion of the total.…”

Section: Finite Change Analysismentioning

confidence: 99%

Finite change analysis of glycolytic intermediates in tuber tissue of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase

Thomas

Mooney²,

Burrell³

et al. 1997

Biochemical Journal

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Genetically engineered organisms overexpressing phosphofructokinase (PFK), a supposed 'regulatory' step of glycolysis, often show little or no measurable change in glycolytic or respiratory flux, although the concentrations of glycolytic intermediates may change. We have used the finite change theory of Metabolic Control Analysis (MCA) to analyse the concentrations of glycolytic metabolites in aged disks of tuber tissue from four lines of transgenic potatoes expressing different amounts of PFK that, under aerobic conditions, showed statistically indistinguishable rates of respiration. The constancy of the metabolites' concentration deviation indices for different increases in PFK expression indicated that the metabolite changes from a graded series, excluding the possibility of anomalous behaviour that might be observed in a single transgenic line. Consequently we were able to use the finite change method to validate the results of an MCA model of tuber glycolysis [Thomas, Mooney, Burrell and Fell (1997) Biochem. J. 322, 119-127]. Furthermore the metabolite changes with PFK activity are evidence that near-equilibrium steps do not transmit increased substrate concentrations down the pathway without attenuation. Our results support the view that flux increase by activation of a single enzyme early in the pathway will, contrary to expectations, be of limited effectiveness in achieving flux increases.

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“…Assay performed according to the reaction described by Kemerer et al [1975], based on conversion of the reaction product, fruc tose-1,6-diphosphate to a-glycerophosphate in the presence of aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase, with the result ing oxidation of NADH.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Inhibitory Effect of Saccharin on Glycolytic Enzymes in Cell-Free Extracts of Streptococcus mutans

Linke

Kohn

1984

Caries Res

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Inhibitory effect of sodium saccharin in concentrations from 10^––5 to 10^––2M was tested on glycolytic enzymes involved in phosphate transfer in cell-free extracts of Streptococcus mutans NCTC 10449. In the presence of 10^––2M sodium saccharin the enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, pyruvate kinase, 3-phosphoglycerate kinase and phosphofructokinase were inhibited 58, 45, ∼16, 12, 3 and 1.4%, respectively. 10^––3–10^––2M sodium chloride produced little if any inhibition of the glycolytic enzymes tested.

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[16] Phosphofruktokinase from Escherichia coli

Cited by 15 publications

References 9 publications

Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Finite change analysis of glycolytic intermediates in tuber tissue of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase

Inhibitory Effect of Saccharin on Glycolytic Enzymes in Cell-Free Extracts of Streptococcus mutans

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