Liver Phosphofructokinase Research Articles

Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo.

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.

Regulation of rat liver phosphofructokinase levels in Morris hepatomas

The levels of the major liver phosphofructokinase isozyme (PFK-L 2) and the PFK regulatory factor were measured in adult and fetal liver as well as Morris hepatomas of different differentiation states. The results indicate that both the PFK-L 2 activity and the PFK regulatory factor levels in well and highly differentiated hepatomas are not statistically different from the amounts found in adult liver. In the poorly differentiated hepatomas and fetal liver, the levels of both PFK-L 2 and PFK regulatory factor are approximately 2 and 3 fold greater, respectively, than what was found in adult liver.

Phosphofructokinase in the rat testis during maturation and following heat treatment in vitro and in vivo.

Activity of phosphofructokinase (PFK) was measured in testes of rats during sexual maturation to determine whether the enzyme was concentrated in spermatogenic or interstitial elements. PFK was also assayed following incubation at 29-38#{176} C in vitro and following short term cryptorchidism to measure the lability of the enzyme to different temperatures. Specific activity (s.a.) of PFK decreased (P<0.05) from 11.81 ± 0.37 units/mg protein in testes from 20-day-old rats to 7.19 ± 0.18 units/mg at 30 days and 6.00 ± 0.14 units/mg at 40 days of age. Activity leveled off at 6.406.50 units until 60 days, then increased to 8.01 ± 0.43 at 120 days of age. Cryptorchidism in adult rats reduced s.a. of PFK to 93% of controls (P<0.05) in 12 h and to 80% of controls (P<0.05) in 48 h. Incubation for 2 h in vitro resulted in higher (P<0.05) s.a. of PFK in testes incubated at 32#{176}C than at 29, 35 or 38#{176}C, but s.a. of PFK in kidney slices was unaffected by incubation temperature. Liver PFK activity decreased with increasing temperature. The results suggest that testic� ular PFK is more concentrated in interstitial tissues than in spermatogenic elements, but that PFK activity in the latter is reduced by abdominal temperatures.

Regulation of Liver Phosphofructokinase by Phosphorylation-Dephosphorylation

Regulation of Liver Phosphofructokinase by Phosphorylation-Dephosphorylation

Purification and properties of liver phosphofructokinase from normal and obese mice

1. 1. Liver phosphofructokinase has been purified from normal and obese mice to a final specific activity of 50–70 units/mg. No difference between the specific activity of the enzyme from either type of mouse was observed. 2. 2. The regulatory properties of the purified enzyme was examined. No physiologically relevant differences in inhibition by ATP or citrate or activation by AMP were noted between normal and obese mice.

Phosphofructokinase Isozymes from Human Organs and Blood Cells

Phosphofructokinase (PFK) isozymes of blood cells and some human tissues were studied by starch gel electrophoresis and immunoprecipitation by anti-muscle and anti-erythrocyte PFK sera. PFK from muscle, heart, brain and placenta were totally precipitated by both antisera. PFK from blood cells (erythrocytes, lymphocytes, granulocytes, platelets) were precipitated more strongly by anti-erythrocyte PFK serum than by anti-muscle PFK serum. Liver, kidney and monoblast PFK were slightly precipitated by both antisera. From the electrophoretic patterns and the immunoprecipitation curves we may conclude that muscle contains the homotetrameric M4 forms; platelet, liver and kidney the homotetrameric E4 form, and blood cells the M-E hybrids. Monoblasts probably contain a E4 type PFK precursor, and heart, placenta and brain, a modified M4 type PFK. Other isozymes, unrelated with muscle and erythrocyte, were revealed in liver and kidney.

Purification and properties of monkey liver phosphofructokinase

1. 1. Phosphofructokinase (EC 2.7.1.11) was purified from monkey liver. 2. 2. The enzyme was inhibited at high concentrations of ATP. This inhibition was relieved by cyclic AMP or raised concentrations of F6P and/or MgCl 2. 3. 3. At pH 7.0 the kinetics were found to be hyperbolic with sub-inhibitory levels of ATP, and sigmoidal with respect to F6P. 4. 4. Plots of activity vs MgCl 2 concentration were hyperbolic at low concentrations of ATP but biphasic at a high level of ATP (2 mM). 5. 5. The properties of the enzyme appear to be qualitatively similar to those of other mammalian phosphofructokinases but show some quantitative differences.

Turnover of hepatic phosphofructokinase in normal and diabetic rats. Role of insulin and peptide stabilizing factor.

Earlier work demonstrated that the activity of liver phosphofructokinase (PFK-L2) and immunoreactive PFK-L2 were decreased in diabetic rats and increased to normal or super-normal amounts following insulin treatment (Dunaway, G.A., and Weber, G., (1974) Arch. Biochem. Biophys. 162, 629-637). This report indicates that the decrease in levels of PFK-L2 in diabetic rats is a result of an accelerated degradation rate while the synthetic rate remains nearly normal. Following insulin treatment, the rate of PFK-L2 synthesis is enhanced 2-fold, whereas the rate of degradation appears to be greatly diminished. An inverse relationship is shown to exist between the PFK-L2 levels and the rates of PFK-L2 degradation, suggesting that the levels of PFK-L2 are primarily regulated by degradation rate. In addition, the levels of the PFK-L2 peptide stabilizing factor are inversely proportional to rates of PFK-L2 degradation. These results indicate that insulin mediates the rate of degradation of PFK-L2 by controlling the level of the peptide stabilizing factor.

Effects of glucagon on amino acid metabolism in Japanese eels, Anguilla japonica

Actions of glucagon were studied in eels with special reference to amino acid metabolism. A single injection of glucagon in doses of 50–500 μg/kg produced a marked hyperglycemia. The glycemic level was elevated immediately after the injection of glucagon (200 μg/kg) and returned to a normal value 12 hr after the injection. A single injection of glucagon at any dose level from 50 to 500 μg/kg produced no appreciable change in plasma amino nitrogen level. However, repeated injections of the hormone (500 μg/kg/4 hr) during a 12-hr period produced a statistically significant decrease in plasma amino nitrogen level. Repeated injections of glucagon also reduced plasma amino nitrogen level of previously alloxanized fish. Hepatectomy completely abolished the glucagon effect on plasma amino nitrogen in both intact and alloxanized fish. The levels of liver and muscle glycogen and the activities of liver glutamic oxaloacetic transaminase, liver glutamic pyruvic transaminase, liver fructose-1,6-diphosphatase, and liver phosphofructokinase were not affected by glucagon administration. From these results it is concluded that glucagon depressed plasma amino nitrogen level by stimulating hepatic amino acid incorporation from the plasma in eels.

Inhibition of phosphofructokinase by fructose 1,6-diphosphatase in mammalian systems: Protein-protein interaction or fructose 1,6-diphosphate trapping?

The aim of the investigation was to discriminate between a direct inhibitory effect of fructose 1,6-diphosphatase (FDPase) (EC 3.1.3.11) on phosphofructokinase (PFK) (EC 2.7.1.11) and an inhibition of the PFK-catalyzed reaction by removal of FDP. The following results strongly support the second hypothesis, (i) Inhibition of PFK by FDPase directly follows the decrease of the actual concentrations of FDPase. (ii) FDPase has no inhibitory effect as long as the concentration of FDP is kept above 2 μ m. (iii) FDPase has no inhibitory effect when PFK activation is achieved by glucose 1,6-diphosphate, which is not split by FDPase. (iv) FDPase has an inhibitory effect when the activation is achieved by sedoheptulose 1,7-diphosphate, which is split by FDPase. (v) High concentrations of aldolase inhibited the PFK-catalyzed reaction. The degree of inhibition showed a similar correlation with the level of FDP in the assay system when FDPase instead of aldolase was used, (vi) FDP-equilibrated PFK, but not glucose 1,6-diphosphate-equilibrated PFK, is inactivated by FDPase. (vii) Muscle and liver PFK react in the same way with either muscle or liver FDPase.

Pig liver phosphofructokinase: asymmetry properties, proof of rapid association--dissociation equilibria, and effect of temperature and protein concentration on the equilibria.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMetabolic control and structure of glycolytic enzymes. 17. Pig liver phosphofructokinase: asymmetry properties, proof of rapid association-dissociation equilibriums, and effect of temperature and protein concentration on the equilibriumsJohn L. Trujillo and William C. Deal, Jr.Cite this: Biochemistry 1977, 16, 14, 3098–3104Publication Date (Print):July 12, 1977Publication History Published online1 May 2002Published inissue 12 July 1977https://doi.org/10.1021/bi00633a009RIGHTS & PERMISSIONSArticle Views50Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts

A FETAL ISOZYME OF PHOSPHOFRUCTOKINASE IN NEWBORN ERYTHROCYTES

Phosphofructokinase (PFK) exists in isozyme forms in various human tissues. Erythrocytes of newborns have a relative PFK deficiency and an altered rate of glycolysis. PFK in the newborn also shows increased in-vivo lability. Experiments were designed to elucidate the differences between adult and newborn erythrocyte PFK. The purified enzymes from human muscle and liver were separated by chromatography on DEAE-Sephadex A-25 columns equilibrated with 0.1M Tris-PO4 pH 8.0, and elution with a salt gradient. Human muscle and liver PFK eluted as widely separated sharp peaks. Adult erythrocyte PFK (from either crude herolysates or from purified preparations) eluted as 8 single broad peak in a position intermediate between those of the liver and muscle isozymes. Hemolysates from cord blood, examined by this technique, were found to consist of two clearly separated PFK peaks: one chromatographically indistinguishable from adult erythrocyte PFK, and a second peak chromatographically indistinguishable from liver PFK. These data demonstrate that in fetal erythrocytes there is an isozyme presumably identical with liver PFK isozyme, which is probably responsible for the relative deficiency of PFK in newborn erythrocytes.

Phosphofructokinase (Isozymes) Activation by Theophylline or by 3',5' Cyclic AMP Administration

Phosphofructokinase activity of rat thyroid, kidney and muscle can be enhanced by in vivo administration of theophylline or cyclic AMP. This enhancement occurs in the different tissues to a different extent depending on the tissue-specific isozymes. Thyroid and muscle phosphofructokinase which is mainly A-type, is strongly stimulated after administering theophylline in the drinking water over a 8 days period. The kidney enzyme which encompasses about 50% of each A and B type is activated to a lesser extent. Liver phosphofructokinase which is pure B-type shows very little response if any. Comparable results have been obtained with either the muscle or liver enzyme after two to four hours treatment with a single injection of 10 mg/rat of either cyclic AMP or theophylline. This short-term activation could be eliminated by treating the animals with Actinomycin D, 30 min prior to theophylline or cyclic AMP. The possible mechanisms leading to the enhancement of phosphofructokinase activity are discussed.

In vivo and in vitro interconversions of active and inactive forms of phosphofructokinase in rat liver

In vivo and in vitro interconversions of active and inactive forms of phosphofructokinase in rat liver

Purification and physiological role of a peptide stabilizing factor of rat liver phosphofructokinase.

A substance has been purified to apparent homogeneity from rat liver which, as previously reported (Dunaway, G. A., Jr., and Segal, H. L. (1974) Biochem. Biophys. Res. Commun. 56, 689-696), specifically stabilizes the major liver isozyme of phosphofructokinase (PFK-L2) against thermal inactivation and whose level in vivo changes in parallel with and in precedence to that of the enzyme. Molecular weight determinations gave values around 3,500. Evidence for the peptide nature of the factor includes its correspondence with ninhydrin-positive material on gel filtration and paper electrophoresis and its susceptibility to pronase. Electrophoretic behavior indicated at least one free amino group and several carboxyl groups. Amino acid analysis of the peptide yielded only glutamate, glycine, and half-cystine, in equimolar amounts. However, neither GSH nor GSSG have PFK-L2-stabilizing activity. No free sulfhydryl groups were present. Chemical analysis for tryptophan was also negative. The ultraviolet spectrum confirmed the absence of aromatic amino acids. The spectrum exhibited a characteristic peptide peak at 190 nm with no absorbance beyond 240 nm. The factor is unstable to storage in the cold except in the presence of glucose or dithiothreitol. Sucrose, fructose, and GSH were ineffective in this regard. It was slowly denatured by heat or reduced pH even in the presence of glucose. The factor was induced in fasted animals specifically by glucose, of the nutrients tested, and in diabetic animals by insulin. Induction by both glucose and insulin was blocked by cycloheximide and actinomycin. The time course of the glucose induction was the more rapid of the two with a marked overshoot to 3 times normal levels at 12 hours. Increased levels of the factor preceded the increased levels of PFK-L2 brought about by glucose or insulin administration. Native PFK-L2 was inactivated by lysosomal extracts, and this inactivation was strongly inhibited by the peptide factor. These results are in accord with the proposal that the peptide plays a role in regulating PFK-L2 turnover in vivo. The factor also activated the phosphofructokinase-catalyzed reaction by promoting fructose-6-P binding. This effect is analogous to that of AMP on the kinetics of the reaction; however, the factor effect was additive to that of AMP, and the factor did not reverse inhibition by excess ATP as does AMP. We postulate that the stabilizing factor affects an equilibrium between PFK-L2 conformers in favor of one more resistant to lysosomal and thermal inactivation and with greater affinity for fructose-6-P.

Phosphofructokinase and related glycolytic enzyme activities in flounder ( Platichthys flesus, l.) liver

1. 1. Assay of 10 enzymes from flounder liver ( Platichthys flesus) showed highest rates in four dehydrogenases (glucose-6-phosphate, α-glycerophosphate, glyceraldehyde-3-phosphate, and lactate) and two phosphatases (glucose-6-phosphate and fructose-1,6-bis phosphate) and lower rates for three kinases (fructose-6-phosphate, hexose, and pyruvate) and aldolase. 2. 2. Flounder liver phosphofructokinase is a soluble enzyme, inhibited by excess ATP both at pH 6.9 and pH 8.2. At pH 8.2 the inhibition is partially overcome by high levels of fructose-6-phosphate, which shows sigmoid binding kinetics. 3. 3. Fructose-1,6-bis phosphate can be metabolized in the liver extracts to fructose-6-phosphate and inorganic phosphate, or, in the presence of NAD, to a mixture of 3-phosphoglycerate and α-glycerophosphate (via aldolase, α-glycerophosphate and glyceraldehyde 3-phosphate dehydrogenases). The possible significance of these steps to the in vivo carbohydrate metabolism is discussed.

Liver peptide stabilizing factor protects phosphofructokinase against inactivation by fructose-1,6-bisphosphatase

Rabbit liver fructose-1,6-bisphosphatase (FDPase) can reversibly inactivate both rabbit muscle and rat liver phosphofructokinases (PFK) under appropriate conditions. The peptide factor which stabilizes rat liver PFK-L 2 against thermal inactivation has now been found to protect both PFKs from inactivation by FDPase. Assay at high ATP (ca. 3 mM) is necessary to demonstrate these reversible changes. In addition, the activation of FDPase by liver cytosol, by oleate plus cytosol, or by oleate plus muscle PFK is lowere about 50% in the presence of peptide factor. These observations suggest an active participation of the peptide factor in regulation of liver glycolysis and gluconeogenesis.

Distribution of phosphofructokinase isozymes in rabbit, mouse, guinea pig and rat

1. 1. The distribution of phosphofructokinase total activity and isozyme patterns have been compared among rabbit, rat, guinea pig, and mouse tissues. 2. 2. In all species, the patterns were basically similar with PFK-A present as the sole or major form of muscle and heart and with PFK-B the major or sole form of liver and erythrocytes. 3. 3. Electrophoretic patterns indicated the presence of hybrid isozymes in all species. 4. 4. Antisera prepared against purified rabbit muscle and liver phosphofructokinases were capable of binding phosphofructokinases of rat and mouse.

Activation and inactivation of rat liver phosphofructokinase by phosphorylation—dephosphorylation

Activation and inactivation of rat liver phosphofructokinase by phosphorylation—dephosphorylation

Rat Liver Phosphofructokinase: PURIFICATION AND CHARACTERIZATION OF ITS REACTION MECHANISM

Abstract Rat liver phosphofructokinase was purified to homogeneity with a specific activity of 84 units per mg of protein. The sodium dodecyl sulfate-treated purified enzyme migrated as a single band in 5% sodium dodecyl sulfate polyacrylamide gel with an apparent molecular weight of 82,000 for the protomer. Polyacrylamide gradient gel electrophoresis demonstrated that the molecular weight of the purified enzyme was 325,000 to 365,000 indicating that the monomer consists of four protomers. The initial velocity kinetics in the forward and the reverse direction revealed clearly intersecting patterns. The product inhibition kinetics were as follows. Fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at nonsaturating and saturating concentrations of MgATP. In the reverse reaction, fructose-6-P was a competitive inhibitor versus fructose-1,6-P2 at saturating concentrations of MgADP. The noncompetitive inhibition of fructose-1,6-P2 versus MgATP was completely overcome by saturating concentrations of fructose-6-P. MgADP was a noncompetitive inhibitor versus MgATP both at nonsaturating and saturating concentrations of fructose-6-P. MgADP exhibited also noncompetitive inhibition versus fructose-6-P at nonsaturating concentrations of MgATP but the inhibition became uncompetitive at saturating concentrations of MgATP. The results are in accordance with an ordered Bi Bi reaction mechanism where fructose-6-P is the first, MgATP the second substrate to bind, and MgADP the first and fructose-1,6-P2 the second product to be released.