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PubMed1983

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver.

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.

Animals↗
PubMed1983

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases.

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.

Adenosine Monophosphate↗
PubMed1983

Interaction of ribulose bisphosphate carboxylase/oxygenase with 2-carboxyhexitol 1,6-bisphosphates.

2-C-Carboxy-D-glucitol 1,6-bisphosphate (CGBP) and 2-C-carboxy-D-mannitol 1,6-bisphosphate (CMBP) have been synthesized, isolated, and the structures of these compounds and the derived lactones elucidated by NMR spectroscopy and periodate oxidation. Both carboxyhexitol bisphosphates, which are homologs of the transition state analog 2-C-carboxy-D-arabinitol 1,5-bisphosphate, exhibit competitive inhibiton of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.9) isolated from spinach (Spinacia oleracea), with respect to ribulose 1,5-bisphosphate. CMBP was a more potent inhibitor (100-fold) displaying an inhibition constant (Ki at pH 8.0 and 30 degrees C) of 1-2 microM with enzymes from spinach, barley (Hordeum vulgare), and Chromatium vinosum. In contrast the Rhodospirillum rubrum enzyme was inhibited about 40-fold more weakly (Ki = 53 microM at pH 8.0 and 30 degrees C). Both CGBP and CMBP potentiated activation of RuBP carboxylase from spinach and R. rubrum.

Binding Sites↗
PubMed1984

Purification of the phosphofructokinase regulatory factors.

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.

Animals↗
PubMed1984

Properties of the phosphofructokinase regulatory factors.

The work presented herein describes many of the physiological properties of the phosphofructokinase regulatory factors. Factor activity can be separated into two discrete fractions, which were designated factor A and factor B, based on their respective charges. A preparation containing both factor A and factor B did not protect the following key carbohydrate-metabolizing enzymes from thermal inactivation: glucokinase, glucose-6-phosphatase (solubilized or nonsolubilized forms), pyruvate kinase, glucose-6-P dehydrogenase, muscle-type phosphofructokinase, or the minor liver phosphofructokinase isozyme. Factor activity in this sample was found to be Pronase sensitive, irreversibly precipitated by trichloroacetic acid, reversibly precipitated by adjusting the sample to a pH of 3.0, and stable to heating at 98 degrees C for 20 min. Distribution studies indicated that factor activity was found only in the soluble cell fraction and not in the mitochondrial or nuclear fractions. Factor activity was retained by 12,000-14,000 molecular weight cut-off (MWCO) dialysis tubing, and not retained by 50,000 MWCO dialysis tubing. These studies indicate that fructose-2,6-P2, calmodulin, or insulin-generated mediator are not associated with factor activity. Although fructose-2,6-P2 did not, both factor preparations protect the major liver phosphofructokinase isozyme (liver PFK) from inactivation by lysosomal extracts. In the diabetic rat, the activities of both factors are greatly reduced but return to near normal levels after 48 h of insulin administration. These data suggest that factor B had little or no effect on the kinetic properties of liver PFK. However, factor A was a K-type activator with respect to fructose-6-P, increasing both the Km and Ki for ATP, and slightly increasing the Vm.

Amino Acids↗
PubMed1984

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding.

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.

Alanine↗
PubMed1984

Activation of phosphofructokinase from rat tissues by 6-phosphogluconate and fructose 2,6-bisphosphate.

6-Phosphogluconate activates phosphofructokinase from liver, adipose tissue, kidney, and skeletal muscle by decreasing the apparent S0.5 for fructose 6-phosphate without affecting the maximum velocity. The response of phosphofructokinase to 6-phosphogluconate is hyperbolic, with apparent activation constants similar to concentrations of 6-phosphogluconate in tissues. Phosphofructokinase from these tissues is also activated by fructose 2,6-bisphosphate, but the apparent activation constants are much less than the concentrations of fructose 2,6-bisphosphate in tissues. Under most conditions, the effects of 6-phosphogluconate and fructose 2,6-bisphosphate are additive. However, with low concentrations of fructose 6-phosphate there is synergism between the effectors. Whereas fructose 2,6-bisphosphate overcomes the inhibition of phosphofructokinase by high concentrations of ATP, 6-phosphogluconate does not. Thus, the effectors probably act at different sites on the enzyme. The relative effect of 6-phosphogluconate is much greater on phosphofructokinase from the lipogenic tissues, adipose, and liver, than it is on the enzyme from kidney or skeletal muscle. Thus, the influence of 6-phosphogluconate on phosphofructokinase, which could coordinate the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis, may be important for lipogenesis.

Adenine Nucleotides↗
PubMed1987

Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver.

Mammalian and yeast hexokinases were found to be reversibly inhibited by fructose 2,6-bisphosphate, an effect requiring the presence of a cytosolic protein factor. Experimental evidence suggests that this factor (inhibitor) is a regulatory protein, the interactions of which with hexokinases are modulated by fructose 2,6-bisphosphate. The Vmax of hexokinase D was decreased, and no changes on other kinetic parameters were observed. The inhibitor was present in fresh liver cytosol filtered through Sephadex G-25 and was partially isolated by negative absorption on DEAE-cellulose followed by ammonium sulfate fractionation. The inhibitor was also present in brain and kidney, but not in muscle. A molecular mass of 200,000 was determined by gel filtration. The inhibition was dependent on the concentrations of both the inhibitory protein and fructose 2,6-bisphosphate. No delay in fructose 2,6-bisphosphate inhibition was observed. Several other hexose phosphates were tested and were not effective. In the presence of amounts of inhibitor sufficient to produce complete inhibition of hexokinase D, the concentration of fructose 2,6-bisphosphate required to produce 50% inhibition was about 0.5 microM. The inhibitor was unstable and was stabilized by the presence of fructose 2,6-bisphosphate.

Animals↗
PubMed1988

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts.

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.

Animals↗
PubMed1975

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts.

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinites for fructose diphosphate and Mg-2+ and were similarly activated by EDTA, dithiothreitol and cystamine. Mg-2+, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.

Chloroplasts↗
PubMed1976

Photosynthesis in a reconstituted chloroplast system from spinach. Some factors affecting CO2-dependent oxygen evolution with fructose-1,6-bisphosphate as substrate.

When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, 14CO2 fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O2 evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway.

Carbon Dioxide↗