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6 Phosphofructokinase

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Sumiko Inouye – One of the best experts on this subject based on the ideXlab platform.

Stephen B. Smith – One of the best experts on this subject based on the ideXlab platform.

  • postmortem regulation of glycolysis by 6 Phosphofructokinase in bovine m sternocephalicus pars mandibularis
    Meat Science, 2005
    Co-Authors: Ryan D. Rhoades, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Andy D King, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P<0.001) from 0.84μmol/g at d 0 to 6.54μmol/g at d 4. Fructose6phosphate and glucose-6phosphate increased (P<0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33μmol/g) and was elevated to 45.9μmol/g by d 4. In the second experiment, conversion of [U-(14)C]glucose to lactate, glycogen, and CO(2) was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U-(14)C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123nmol glucose utilized per 100mg muscle per h; P=0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO(2) in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.

  • Postmortem regulation of glycolysis by 6Phosphofructokinase in bovine M. Sternocephalicus pars mandibularis
    Meat science, 2005
    Co-Authors: Ryan D. Rhoades, D. Andy King, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P

Hirofumi Nariya – One of the best experts on this subject based on the ideXlab platform.

  • modulating factors for the pkn4 kinase cascade in regulating 6 Phosphofructokinase in myxococcus xanthus
    Molecular Microbiology, 2005
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus, a Gram-negative developmental bacterium, contains a large number of protein Ser/Thr kinases (PSTKs). Among these PSTKs, Pkn4 has been shown to be 6Phosphofructokinase (PFK) kinase. PFK associates with the regulatory domain of Pkn4 (Pkn4RD) and is activated by Pkn4-mediated phosphorylation. The activation of PFK is required to consume glycogen accumulated during early development and is essential for efficient sporulation. Using the yeast two-hybrid screen, we identified three new factors, MkapA, MkapB and MkapC, that interact with Pkn4 and each contains well-known protein–protein interaction domains. MkapB contains eight tandem repeats of the TPR (tetratrico peptide repeat) domain and its interaction with Pkn4RD was phosphorylation-dependent. MkapB remained associated with Pkn4RD. As a result, Pkn4 did not interact with PFK and its activation was inhibited. While deletion of the pfk-pkn4 operon did not inhibit fruiting body formation, the spore yield was low. In contrast, a mkapB deletion mutant exhibited a 24 h delay in fruiting body formation, accumulated less glycogen in the stationary phase and gave rise to 3.2% spore formation as opposed to 100% attained with DZF1. In addition to Pkn4, MkapA associated with other membrane-associated PSTKs, Pkn1, Pkn2, Pkn8 and Pkn9, while MkapB associated with Pkn8 and Pkn9, and MkapC with Pkn8. These results indicate that there are complex PSTK networks in M. xanthus that share common modulating factors.

  • An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4-activated 6Phosphofructokinase.
    Molecular microbiology, 2003
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary 6Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7-fold upon phosphorylation at Thr-226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk–pkn4 deletion strain accumulated glycogen at a higher level than the wild-type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk–pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr-226-Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.

  • Activation of 6Phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus.
    Molecular microbiology, 2002
    Co-Authors: Hirofumi Nariya, Sumiko Inouye
    Abstract:

    Summary Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6Phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.

Ryan D. Rhoades – One of the best experts on this subject based on the ideXlab platform.

  • postmortem regulation of glycolysis by 6 Phosphofructokinase in bovine m sternocephalicus pars mandibularis
    Meat Science, 2005
    Co-Authors: Ryan D. Rhoades, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Andy D King, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P<0.001) from 0.84μmol/g at d 0 to 6.54μmol/g at d 4. Fructose-6-phosphate and glucose-6-phosphate increased (P<0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33μmol/g) and was elevated to 45.9μmol/g by d 4. In the second experiment, conversion of [U-(14)C]glucose to lactate, glycogen, and CO(2) was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U-(14)C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123nmol glucose utilized per 100mg muscle per h; P=0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO(2) in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.

  • Postmortem regulation of glycolysis by 6Phosphofructokinase in bovine M. Sternocephalicus pars mandibularis
    Meat science, 2005
    Co-Authors: Ryan D. Rhoades, D. Andy King, Blaine E. Jenschke, Jason M. Behrends, Teresa S. Hively, Stephen B. Smith
    Abstract:

    This experiment addressed the hypothesis that 6Phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P

Peter Schonheit – One of the best experts on this subject based on the ideXlab platform.