Showing papers on "Pyruvate kinase published in 1991"
TL;DR: Results show that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.
Abstract: The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the “sink” regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 μmol photons · m−2 · s−1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO2, less marked in saturating irradiance and saturating CO2, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.
250 citations
TL;DR: It is concluded that changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.
Abstract: Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the “sink” regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO2. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.
226 citations
TL;DR: The results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors.
Abstract: Relative levels of many individual proteins in Escherichia coli HB101 strains with 0, 37, 56, and 240 plasmids per chromosome were determined by computer image analysis of two-dimensional gel electrophoresis patterns The plasmids investigated had very similar sequences except for small domains encoding the repressor of plasmid replication At the intermediate plasmid copy number of 56, levels of several of the TCA cycle enzymes (oxoglutarate dehydrogenase complex, succinate thiokinase, and succinate dehydrogenase) as well as in aspartate transcarbamoylase increased At a plasmid copy number of 240, higher amounts of PEP carboxylase as well as several of the heat shock proteins were observed Furthermore, at high plasmid levels, significant decreases occurred in growth rate, pyruvate kinase I, pyruvate dehydrogenase complex, unadenylated glutamine synthetase, aspartate transcarbamoylase as well as in several of the proteins involved in translation Decreases in ribosome content as well as in the free 30S and 50S ribosomal subunit pool fractions were also observed in separate analyses These results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors
175 citations
TL;DR: Determination of Fru-1,6-P2 indicated that its intracellular concentration decreased concomitantly with the reduction in glucose concentration in the medium, indicating that monomer-tetramer inter-conversion is a major in vivo cellular regulatory mechanism in response to changes in the extracellular glucose concentration.
161 citations
TL;DR: During Pi limitation, PEP carboxylase and PEP phosphatase may function to "bypass" the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.
Abstract: The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to “bypass” the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.
151 citations
TL;DR: The 101-base pair fragment from -197 to -96 of the L-PK gene can confer carbohydrate regulation when fused in either orientation to the heterologous thymidine kinase promoter, thus defining a carbohydrate response element in this region.
145 citations
TL;DR: The view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes is supported.
105 citations
TL;DR: The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied and the relative levels of each gly colytic mRNA were compared during exponential growth on glucose or lactate, found to be induced to differing extents by glucose.
Abstract: The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied. When the relative levels of each glycolytic mRNA were compared during exponential growth on glucose or lactate, the various glycolytic mRNAs were found to be induced to differing extents by glucose. No significant differences in the stabilities of the PFK2, PGK1, PYK1, or PDC1 mRNAs during growth on glucose or lactate were observed. PYK::lacZ and PGK::lacZ fusions were integrated independently into the yeast genome at the ura3 locus. The manner in which these fusions were differentially regulated in response to carbon source was similar to that of their respective wild-type loci. Therefore, the regulation of glycolytic mRNA levels is mediated at the transcriptional level. When the mRNAs are ordered with respect to the glycolytic pathway, two peaks of maximal induction are observed at phosphofructokinase and pyruvate kinase. These enzymes (i) catalyze the two essentially irreversible steps on the pathway, (ii) are the two glycolytic enzymes that are circumvented during gluconeogenesis and hence are specific to glycolysis, and (iii) are encoded by mRNAs that we have shown previously to be coregulated at the translational level in S. cerevisiae (P. A. Moore, A. J. Bettany, and A. J. P. Brown, NATO ASI Ser. Ser. H Cell Biol. 49:421-432, 1990). This differential regulation of glycolytic mRNA levels might therefore have a significant influence upon glycolytic flux in S. cerevisiae.
102 citations
TL;DR: The results suggest that the primary action of benzoic acid is to cause a general energy loss, i.e., ATP depletion, in the preservative-resistant yeast Zygosaccharomyces bailii.
Abstract: The effects of benzoic acid in the preservative-resistant yeast Zygosaccharomyces bailii were studied. At concentrations of benzoic acid up to 4 mM, fermentation was stimulated and only low levels of benzoate were accumulated. Near the MIC (10 mM), fermentation was inhibited, ATP levels declined, and benzoate was accumulated to relatively higher levels. Intracellular pH was reduced but not greatly. Changes in the levels of metabolites at different external benzoic acid levels showed that glycolysis was limited at pyruvate kinase and glyceraldehyde dehydrogenase-phosphoglycerate kinase steps. Inhibition of phosphofructokinase and several other glycolytic enzymes was not responsible for the inhibition of fermentation. Instead, the results suggest that the primary action of benzoic acid in Z. bailii is to cause a general energy loss, i.e., ATP depletion.
95 citations
TL;DR: Results indicated that p58-M2 has intrinsic kinase activity, and the order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity.
Abstract: We have recently shown that the cytosolic thyroid hormone binding protein (p58-M2) in human epidermoid carcinoma A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2). To characterize further the molecular properties of p58-M2, we overexpressed p58-M2 in Escherichia coli and purified it to homogeneity. At 22 degrees C, the monomeric p58-M2, exhibited kinase activity with an apparent Vmax of 22 +/- 9 units/mg. The Km for adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) are 3.85 +/- 2.4 and 1.55 +/- 0.73 mM, respectively. Upon activation by fructose 1,6-bisphosphate (Fru-1,6-P2), Vmax and Km for ADP and PEP were changed to 490 +/- 27 units/mg and 0.63 +/- 0.09 and 0.13 +/- 0.01 mM, respectively. These results indicated that p58-M2 has intrinsic kinase activity. Analysis of the molecular size indicated that the activation of p58-M2, by Fru-1,6-P2 resulted in the association of the monomeric p58-M2 to the tetrameric PKM2. p58-M2 bound to 3,3',5-triiodo-L-thyronine (T3) (Ka = 1.7 x 10(7) M-1) and exhibited analogue specificity, whereas PKM2 did not bind thyroid hormone. The order of binding affinity was L-T3 greater than L-thyroxine greater than 3,3',5-triiodothyropropionic acid greater than 3'-isopropyl-3,5-triiodo-L-thyronine greater than 3'5',3-triiodo-L-thyronine. Binding of T3 and its analogues resulted in the inhibition of the kinase activity of p58-M2. The order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
90 citations
TL;DR: It is concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.
Abstract: cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with hereditary hemolytic anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.
TL;DR: Results of metabolic, enzymic, and molecular biologic experiments were each consistent with an intramitochondrial site(s) of down-regulation in islets cultured at low glucose, indicating that regulation by glucose was at transcription of genes coding for some mitochondrial enzymes.
TL;DR: Application of the recently developed quantitative theory of metabolic control of branched pathways provides a hypothesis to account for the high rate of both glycolysis and glutaminolysis in lymphocytes, macrophages and, in particular, in tumor cells.
TL;DR: Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined and alcohol dehydrogensase II was identified as a major stress protein.
Abstract: The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45 degrees C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins. Images
TL;DR: The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome.
Abstract: In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes We have cloned and characterized one of the alleles Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids The gene sequences differ at 15 positions, resulting in five amino acid substitutions The calculated molecular masses of the polypeptides are 54,378 Da and 54,363 Da These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57-59 kDa However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41-51% The conserved residues are not equally distributed over the polypeptide The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable The cytosolically located pyruvate kinase of T brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only +3
TL;DR: Results confirm that transduction of the insulin signal occurs via distinct pathways; one of these pathways could involve a secondary insulin-dependent modification of metabolite concentration, whereas other pathways could be more directly related to the activity(ies) of the occupied insulin receptor.
TL;DR: It is concluded that inhibition of gluconeogenesis by TCDD previously demonstrated in vivo is probably due to decreased activities of PEPCK and PC, and the data support the prevailing view that PEPCk and PC are rate-determining enzymes in gluc oneogenesis.
Abstract: Male Sprague-Dawley rats (240–245 g) were dosed ip with 5, 15, 25, or 125 μg/kg -,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. Ad libitumfed and pair-fed controls received vehicle (4 ml/kg) alone. Two or 8 days after dosing five rats of each group were sacrificed, their livers removed and assayed for the activities of three gluconeogenic enzymes [phosphoenol-pyruvate carboxykinase (PEPCK; EC 4.1.1.32), pyruvate carboxylase (PC; EC 6.4.1.1.), and glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9)], and one glycolytic enzyme [pyruvate kinase (PK; EC 2.7.1.40)] by established procedures. The activity of PK was not affected by TCDD at either time point. The activity of G-6-Pase tended to be decreased in TCDD-treated animals, as compared to pair-fed controls, but the decrease was variable without an apparent dose-response. The activity of PEPCK was significantly decreased 2 days after dosing, but a clear dose-response was apparent only at the 8-day time point. Maximum loss of activity at the highest dose was 56% below pair-fed control levels. PC activity was slightly decreased 2 days after TCDD treatment and displayed statistically significant, dose-dependent reduction by 8 days after dosing with a 49% loss of enzyme activity after the highest dose. It is concluded that inhibition of gluconeogenesis by TCDD previously demonstrated in vivo is probably due to decreased activities of PEPCK and PC. The data also support the prevailing view that PEPCK and PC are rate-determining enzymes in gluconeogenesis.
TL;DR: The results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus, and suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidativeosphorylation.
TL;DR: It is argued that PFP probably has a glycolytic function in potato tubers, supplementing the rather low ATP-dependent phosphofructokinase activity, allowing PFP to operate in the reverse direction, and generate inorganic pyrophosphate which is needed for sucrose mobilisation.
TL;DR: It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.
Abstract: The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.
TL;DR: Both enzyme variants show a markedly reduced specific activity and thermolability in pyruvate kinase (PK) deficiency.
TL;DR: Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours, but in mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity.
TL;DR: The activity of glycolytic enzymes GAPDH and PK and the tricarboxylic acid (TCA) cycle enzyme ICDH was low compared to that in non-dormant buds when buds were dormant.
Abstract: The activity of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphate gluconate dehydrogenase (5PGDH), isocitrate dehydrogenase (ICDH), pyruvate kinase (PK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were studied in apple (Malus domestica Borkh.) buds during dormancy and thidiazuron-induced budbreak. When buds were dormant, the activity of the glycolytic enzymes GAPDH and PK and the tricarboxylic acid (TCA) cycle enzyme ICDH was low compared to that in non-dormant buds. The activity of these enzymes increased during budbreak, peaked when buds were in the green tip stage just before the start of rapid expansion (at 8 days after thidiazuron treatment), and declined thereafter (...)
TL;DR: The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%.
Abstract: cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.
TL;DR: The bioactivities of RRR-α-, β-, γ-, and δ-to-copherol (T) and R-α-tocotrienol (Rα-TT) were determined in rat resorption-gestation tests.
TL;DR: Investigation of the effect of increased fluxes of H+ across the plasmalemma on glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum L shows that the primary sites for regulation of gly colysis are pyruvate kinase and phosphoenolpyruVate carboxylase, respectively.
Abstract: The present experiments were carried out to investigate the effect of increased fluxes of H+ across the plasmalemma on glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum L. (1) Increased H+ influx was produced by adding glucose, 6-deoxyglucose, 2-deoxyglucose, or sodium fluoride. The net influx decreased to zero after 3 min. This recovery was accompanied by an increase in the rate of O2 uptake, but not of dark CO2 fixation. When glucose or fluoride were added, the increase of O2 uptake occurred without a decrease in the ATP/ADP ratio, and was large enough to provide the ATP that would be needed for compensatory H+ extrusion via the plasmalemma H+-ATPase. When 2-deoxyglucose was added, the rise of respiration was restricted by sequestration of phosphate and depletion of phosphorylated metabolites, the ATP/ADP ratio declined, and a slow net H+ influx started again after 4 min. (2) Alkalinisation of the medium to induce an H+ efflux resulted in rapid activation of dark CO2 fixation, but not of O2-uptake. (3) A stimulation of respiration or dark CO2 fixation was always accompanied by a decrease of phosphoenolpyruvate. This shows that the primary sites for regulation of glycolysis are pyruvate kinase and phosphoenolpyruvate carboxylase, respectively. (4) There was no consistent relation between glycolytic flux and triose-phosphates or hexose-phosphates. This shows that the reactions involved in carbohydrate mobilisation and the conversion of hexose-phosphates to triose-phosphates only have a secondary role in stimulation of glycolysis. (5) Phosphofructokinase will be stimulated as a consequence of the decrease in phosphoenolpyruvate. (6) The increase in glycolytic flux occurred independently of (in the case of 2-deoxyglucose and fluoride), or before (in the case of glucose), any increase of fructose-2,6-bisphosphate. When fructose-2,6-bisphosphate did increase (after supplying glucose), this was accompanied by an increase of triose-phosphate and fructose-1,6-bisphosphate, which otherwise remained very low. It is argued that fructose-2,6-bisphosphate increases as a consequence of the decrease of glycerate-3-phosphate, a known inhibitor of the synthesis of this regulator metabolite. However, activation of pyrophosphate fructose-6-phosphate phosphotransferase by fructose-2,6-bisphosphate does not play an obligatory role in the stimulation of glycolysis.
TL;DR: An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
Abstract: In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
01 Dec 1991
TL;DR: In the liver, with the exception of fructose-diphosphatase, the return to basic values was much quicker than in intestine, and 7 enzymes out of 8 it was realized in 9 days or less, when a high fructose diet gives way to a normal one.
Abstract: In this research on metabolic effects of a high fructose diet, we studied the duration of these effects by measuring the specific activity of 8 enzymes stimulated by such a diet, on days, 0, 3, 6, 9, 15, after the return to a normal diet. In the intestinal mucosa, ketohexokinase, aldolase, triokinase, fructose-diphosphatase, and glucose-6-phosphatase specific activities were still entirely or partially stimulated on the 15th day after return to the standard diet. The stimulation of glucose-6-phosphatase and pyruvate kinase specific activities stopped quickly. In the liver, with the exception of fructose-diphosphatase, the return to basic values was much quicker than in intestine. In 7 enzymes out of 8 it was realized in 9 days or less. When a high fructose diet gives way to a normal one, return to basic values comes so much the quicker as activation has needed longer to appear.
TL;DR: The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose and a possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.
TL;DR: MP and MHP have the properties to act in vivo as compatible osmotica, and increased, in vitro, the thermal stability of PK from both plant and animal sources, and protected PK (Zea mays) from salt inhibition at two substrate levels.
Abstract: The nitrogenous compounds N-methyl-L-proline (MP), trans-4-hydroxy-N-methyl-L-proline (MHP) and trigonelline (T), which undergo stress-induced accumulation in some Australian plants, were analysed and compared with proline (P) and glycinebetaine (B) for possible protective roles. The activity of pyruvate kinase (PK), prepared from Zea mays leaves and rabbit muscle, was unaffected even in the presence of 750 mM of the proline analogues. Thus, MP and MHP, like P and B, have the properties to act in vivo as compatible osmotica. T was not as compatible, decreasing enzyme activity 20% at 0.5 M. Like P and B, however, MP, MHP and T all also exhibited protective properties. They increased, in vitro, the thermal stability of PK from both plant and animal sources, and they protected PK (Zea mays) from salt inhibition at two substrate levels. The effect of salt on PK (Zea mays) was substrate dependent; at low phosphoenolpyruvate (PEP) levels, salt inhibited the enzyme activity, while salt effects were less severe in the presence of higher substrate levels. In the presence of high NaCl concentrations, the protective effects of high substrate levels and the compatible solutes seem to be additive. The Km (PEP) value of the plant PK increased in the presence of salt but the effect was ameliorated by the compatible solute MHP.