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PubMed2004

Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity.

A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The Km values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mm for fructose 6-phosphate and 1.2 mm for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMP-dependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.

Acetylglucosamine↗
PubMed2004

6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia.

The up-regulation of glycolysis to enhance the production of energy under reduced pO(2) is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5'-flanking region. Extensive analysis of the 5' pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1alpha null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.

Animals↗
PubMed2005

High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit.

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.

Animals↗
PubMed2005

Crystal structure of the hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): a possible new target for cancer therapy.

The hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) plays a crucial role in the progression of cancerous cells by enabling their glycolytic pathways even under severe hypoxic conditions. To understand its structural architecture and to provide a molecular scaffold for the design of new cancer therapeutics, the crystal structure of the human form was determined. The structure at 2.1 A resolution shows that the overall folding and functional dimerization are very similar to those of the liver (PFKFB1) and testis (PFKFB4) forms, as expected from sequence homology. However, in this structure, the N-terminal regulatory domain is revealed for the first time among the PFKFB isoforms. With a beta-hairpin structure, the N terminus interacts with the 2-Pase domain to secure binding of fructose-6-phosphate to the active pocket, slowing down the release of fructose-6-phosphate from the phosphoenzyme intermediate product complex. The C-terminal regulatory domain is mostly disordered, leaving the active pocket of the fructose-2,6-bisphosphatase domain wide open. The active pocket of the 6-phosphofructo-2-kinase domain has a more rigid conformation, allowing independent bindings of substrates, fructose-6-phosphate and ATP, with higher affinities than other isoforms. Intriguingly, the structure shows an EDTA molecule bound to the fructose-6-phosphate site of the 6-phosphofructo-2-kinase active pocket despite its unfavorable liganding concentration, suggesting a high affinity. EDTA is not removable from the site with fructose-6-P alone but is with both ATP and fructose-6-P or with fructose-2,6-bisphosphate. This finding suggests that a molecule in which EDTA is covalently linked to ADP is a good starting molecule for the development of new cancer-therapeutic molecules.

Adenosine Triphosphate↗
PubMed2005

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter by nuclear receptors liver receptor homologue-1 and small heterodimer partner: a mechanism for differential regulation of cholesterol synthesis and uptake.

Cholesterol homeostasis in mammals involves pathways for biosynthesis, cellular uptake, and hepatic conversion to bile acids. Key genes for all three pathways are regulated by negative feedback control. Uptake and biosynthesis are directly regulated by cholesterol through its inhibition of the proteolytic activation of the sterol regulatory element binding proteins. The conversion of cholesterol into bile acids in the liver is regulated through the bile acid-dependent induction of the negatively acting small heterodimer partner nuclear receptor. In this report, we have shown that the small heterodimer partner also directly regulates cholesterol biosynthesis through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase but has no effect on low density lipoprotein receptor expression. This has significant metabolic significance, as it provides both a mechanism to independently regulate cholesterol synthesis from uptake (an essential regulatory feature known to occur in vivo) and a pathway for direct regulation of cholesterol biosynthesis by bile acids. This latter feature ensures that the early phase of bile acid synthesis (pre-cholesterol) is in metabolic communication with the later stages of the pathway to properly regulate whole pathway flux. This highlights an important regulatory feature that is shared with other key branched, multienzyme pathways, such as glycolysis, where pathway outflow through pyruvate kinase is regulated by the concentration of a key early intermediate, fructose 1,6-bisphosphate.

Animals↗
PubMed2000

Regulation of phosphofructokinase-1 on submandibular salivary glands of rats after isoproterenol administration.

The purpose of this investigation was to study the effect of isoproterenol (IPR) treatment on the regulation of phosphofructokinase-1 of submandibular salivary glands of rats. The animals were divided into control and experimental groups. In the first set of experiments, the rats received 5 mg of IPR/kg b.w. and were sacrificed at 24 hours after 1, 2, 3 and 4 doses. The content of fructose-2,6-bisphosphate (Fru-2,6-P(2)) and the activity of 6-phosphofructo-2-kinase (PFK-2) (active and total) were determined. The Fru-2,6-P(2) content was found to be reduced and the activity of PFK-2 (active and total) showed differences from the control. The active/total ratio, was higher for the group of one dose sacrificed 12 hours after the agonist injection as compared to the control. In the other groups, there were reductions which varied from 25 to 33%. In the second set of the experiment, the animals were injected with 23.0 mg of IPR/kg b.w. and were sacrificed from 5 up to 720 minutes after the administration of the agonist. After the sacrifice, salivary gland samples were analyzed for Fru-2,6-P(2). Again, a reduction in the metabolite content was observed. Using beta and alpha receptor blockers, it was found that both inhibited only partially the effect of IPR. The purification of PFK-1 up to homogeneity, from submandibular glands of rats which received 5 mg of IPR/mg b.w. as well as from the control, was performed and the Km and state of phosphorylation were determined. Rats from the group sacrificed 12 hours after the injection of the agonist showed the lowest Km for Fru-6-P. Animals which received 3 doses of IPR showed the highest phosphate content/mol of enzyme. Experiments of dephosphorylation of the purified PFK-1 from this latter group revealed that the presence of the phosphate groups influence the kinetic properties of the enzyme.

Animals↗
PubMed1985

Biotin deficiency and susceptibility to fatty liver and kidney syndrome in broiler chicks: reduced 6-phosphofructokinase (EC 2.7.1.11) activity but normal fructose 2,6-bisphosphate content in birds with hepatomegaly.

In two separate experiments, using different strains, broiler chicks were reared on either a commercial-type chick mash (control) or a fatty liver and kidney syndrome (FLKS)-inducing diet. In Expt a, chicks were killed on day 29 and in Expt b, on day 32. Body-weights and liver weights were measured, and values from those given the control ration used to construct a hepatomegaly index by employing a variant of linear discriminant analysis. Application of the index to FLKS birds revealed a statistically significant bimodal distribution of liver size. The birds with enlarged livers (high index) also possessed metabolic abnormalities in that 6-phosphofructokinase (EC 2.7.1.11; PFK-1) activity (measured at low substrate concentration) was depressed despite the presence of normal, or even slightly elevated fructose 2,6-bisphosphate concentration. This indicates the presence of an uncharacterized regulatory mechanism for PFK-1 in FLKS-susceptible birds.

Animals↗
PubMed2005

Effect of acetic acid feeding on the circadian changes in glycogen and metabolites of glucose and lipid in liver and skeletal muscle of rats.

The aim of the present study is to investigate the effect of acetic acid feeding on the circadian changes in glycogen concentration in liver and skeletal muscle. Rats were provided meal once daily (09.00-13.00 hours) for 10 d. On the 11th day, they were either killed immediately or given 9 g diet containing either 0 (control) or 0.7 g/kg-diet acetic acid beginning at 09.00 hours for 4 h, as in the previous regimen. Rats in the fed group were killed at 4, 8 or 24 h after the start of feeding. At 4 h after the start of feeding, the acetic acid group had significantly greater liver and gastrocnemius muscle glycogen concentrations (P<0.05). Also, at this same point, liver xylulose-5-phosphate, a key stimulator of glycolysis, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate in skeletal muscle, which reflects phosphofructokinase-1 activity, and liver malonyl-CoA, an allosteric inhibitor of carnitine palmitoyl-transferase, were significantly lower in the acetic acid group than in the control group (P<0.05). In addition, the acetic acid group had a significantly lower serum lactate concentration and lower ratio of insulin to glucagon than the control group at the same point (P<0.05). We conclude that a diet containing acetic acid may enhance glycogen repletion but not induce supercompensation, a large increase in the glycogen level that is beneficial in improving performance, in liver and skeletal muscle by transitory inhibition of glycolysis. Further, we indicate the possibility of a transient enhancement of fatty acid oxidation in liver by acetic acid feeding.

Acetic Acid↗
PubMed2002

Prevention of alpha-naphthylthiourea-induced pulmonary edema with fructose-1,6-diphosphate.

Neutrophil-derived oxygen free radicals have been implicated in the pathogenesis of noncardiogenic pulmonary edema. Fructose-1,6-diphosphate (FDP) has been shown to inhibit oxygen free radicals production by activated neutrophils. Thus, we investigated whether FDP would attenuate formation of pulmonary edema in anesthetized dogs injected with alpha-naphthylthiourea (ANTU). Hemodynamic studies involved measurements of left ventricular systolic and end-diasystolic pressures (LVSP and LVEDP), pulmonary artery pressure (PaP), heart rate (HR), and cardiac output (CO). Mean wet weight to dry weight ratios of lung tissue samples were calculated. Following baseline measurements, dogs were injected intravenously (IV) with ANTU 5 mg / kg (n = 16) and 10 mg / kg (n = 8) and half of the dogs were randomly selected to receive 75 mg / kg FDP (10%) and subsequent infusion of 7 mg / kg / min. The rest were given 0.9% NaCl in the same manner. Four hours after ANTU administration, the animals were euthanatized. Except for decline in the CO (nonsignificant), no significant changes in systemic hemodynamics within and between the groups were noted. In the FDP group, PaP and pulmonary arteriolar resistance (PaR) remained unchanged. In the saline group, PaP increased from 12.5 +/- 2.44 to 21.8 +/- 3.14 mm Hg (P < .001) and PaR from 166 +/- 29 to 468 +/- 74 dynes. cm / sec(5) (P < .005). During the study LVDEP, PaO(2), PaCO(2), and hematocrit did not change significantly within and between the groups. The lungs mean wet weight to dry weight ratios for the sham-operated dogs were 4.20 +/- 0.41, for the FDP group 4.32 +/- 0.59 and 6.22 +/- 1.37 for the saline group (P < .0005). These data indicate that FDP protected the lung from ANTU-induced injury.

Animals↗
PubMed2001

Modification of human erythrocyte pyruvate kinase by an active site-directed reagent: bromopyruvate.

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.

Adenosine Diphosphate↗
PubMed2000

Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate.

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.

Buffers↗
PubMed2006

Evolutionary analysis of fructose 2,6-bisphosphate metabolism.

Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representatives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bisphosphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions.

Animals↗
PubMed1997

Kinetic properties of cytosolic fructose 1,6-bisphosphatase from grapefruit. Effect of citrate.

cFBP is studied for its affinity to Mg++ and Fru-1,6-P2. The affinity for Mg++ is not very high with a Km of 0.24 +/- 0.01 mM. High concentrations of Mg++ are inhibitory. The saturation curve for Fru-1,6-P2 is hyperbolic with a Km of 0.54 +/- 0.014 microM. The presence of citrate (10 mM) induces a sigmoidal curve, modifying both Vmax and S0.5. Citrate affects the allosteric properties of cFBPase: at low substrate concentration cooperativity becomes negative while at higher concentration it is positive. Addition of higher concentrations of Mg++ shows a synergistic effect with citrate, decreasing of the affinity for Fru-1,6-P2: S0.5 equals 7.6 +/- 0.25 mM, 9.0 +/- 0.86 mM and 21.5 +/- 1.46 mM in presence of 5, 7.5 and 10 mM Mg++, respectively.

Citric Acid↗
PubMed1998

Fru 2,6-P2 and citrate: intracellular distribution in citrus tissues and effect on grapefruit leaf PFP.

Grapefruit leaf PFP was studied for its activation by fructose 2,6-bisphosphate (Fru 2,6-P2) in the forward and reverse reactions. In the glycolytic reaction, a Ka of 4.0 +/- 0.12 nM was obtained. This constant is affected by the presence of increasing concentrations of citrate (1, 5 and 20 nM) with a Ka(citrate) of 4.5 +/- 0.09, 6.9 +/- 0.05 and 38.2 +/- 1.4 respectively. The inhibition mode of citrate is competitive with Fru 2,6-P2, but non-linear in relation of increasing concentrations of the inhibitor. The intracellular distribution and concentration of the key regulatory metabolite Fru 2,6-P2 was further investigated in citrus leaves and juice cells. Fru 2,6-P2 was only found in the cytosol of juice cells. Fru 2,6-P2 was detected under both conditions with higher concentrations found under aerobiosis.

Citric Acid↗
PubMed2004

Fructose-1,6-diphosphate suppresses T-lymphocyte proliferation, promotes apoptosis and inhibits interleukins-1, 6, beta-actin mRNAs, and transcription factors expression.

The overall objective of this study was to determine the role fructose 1,6-diphosphate (FDP), a naturally occurring glycolytic intermediate, plays in activated T-lymphocytes. The hypothesis is twofold. First, we propose that FDP inhibits T cell proliferation to a greater extent than fructose-1-phosphate (F1P), fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P); second, we argue that FDP suppresses immune activation by inhibiting inflammatory cytokine expression, inhibiting expression of key transcription factors, and by inducing apoptosis in immune cells. Rat spleen cells were incubated with concanavalin A (ConA) and increasing concentrations of FDP. Proliferation was determined by tritiated thymidine uptake. FDP inhibited splenocyte proliferation in a dose-related manner while F1P, F6P, M6P demonstrated inhibition only at high concentrations (5000 microg/ml). RNA was harvested from FDP and ConA-treated cells and IL-1 and IL-6 gene expression was analyzed by RT-PCR. IL-1 and IL-6 mRNA expression was completely inhibited at 500-5000 microg/ml FDP. Apoptosis in FDP-treated lymphocytes was determined by DNA fragmentation and flow cytometry. Propidium iodide (PI) staining demonstrated a 39% rate of apoptosis in splenocytes treated with ConA and 5000 microg/ml FDP. Extensive DNA fragmentation was present at 250-5000 microg/ml FDP, and maximal inhibition occurred at 5 microg/ml. F1P, F6P and M6P showed maximal inhibition only at 5000 microg/ml. Nuclear extracts from FDP-treated splenocytes were analyzed by electrophoretic mobility shift assay. ConA activation of NF-kappaB and AP-1 was dramatically inhibited by FDP. Interestingly, beta-actin showed extensive inhibition with FDP and ConA, thus suggesting new possibilities of its being used as a therapeutic modality in arterial injury where the beta-actin, an important cytoskeleton element, plays a very important role. These data indicate that FDP may be a useful immunosuppressive agent. In conclusion, FDP is not only an immunosuppressant but also an anti-inflammatory agent.

Actins↗
PubMed1990

Changes in intracellular pH caused by high K in normal and acidified frog muscle. Relation to metabolic changes.

We examined the effect of depolarization on intracellular pH (pHi) of normal (pHi approximately 7.37) and acidified (pHi 5.90-6.70) frog semitendinosus muscle using microelectrodes. A small bundle was superfused with a Na(+)-free buffered solution (10 mM HEPES, 100% O2, pH 7.35) containing either 2.5 or 25 mM K+. An NH4Cl prepulse was used to lower pHi. At normal pHi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi of approximately 0.2. In contrast, in all 25 acidified bundles pHi rose by 0.1-0.7. The rise was greater the lower the initial pHi. It could be imitated by caffeine and blocked by tetracaine and thus was, most likely, initiated by release of calcium. We ascribed the alkalinization to hydrolysis of phosphocreatine (PCr); 2,4-dinitrofluorobenzene abolished it. Biochemical analysis on fibers at the peak of alkalinization showed PCr to be reduced by one-half, while PCr in normal fibers that had been depolarized for the same period (4-6 min) showed no change. We postulated that low pHi slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-1,6-diphosphate through inhibition of phosphofructokinase (PFK), an enzyme which is known to be highly pH sensitive. Thus PCr hydrolysis would be required to replace much of the hydrolyzed ATP. This postulated effect on PFK is in agreement with the finding that glucose-6-phosphate (in near-equilibrium with fructose-6-phosphate) was increased nearly fivefold in the depolarized acid fibers, but not in the depolarized normal fibers. However, fructose-1,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced bottleneck between the latter two substrates. We measured the intrinsic buffering power, beta, of frog semitendinosus muscle with small pulses of NH4Cl. It was found to vary with pHi according to beta = 144.6 - 17.2 (pHi).

Adenosine Triphosphate↗
PubMed1994

Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism.

The amino 3'-glycosyl phosphotransferase (neo) gene is the selectable marker most widely used in stable transfection or infection protocols. Because the neo gene product has phosphotransferase activity, it might modify the phosphorylation state when introduced in mammalian cells. NIH-3T3 fibroblast cells expressing the neo gene, after either infection with retroviral vectors or transfection with plasmids, showed a 50% reduction in both fructose 2,6-bisphosphate (Fru 2,6-P2) concentration and lactate production compared with control NIH-3T3 cells, indicating that these neo-expressing cells are less glycolytic. In addition, a marked decrease in the levels of mRNA for the procollagen 1 alpha and fibronectin genes was also observed in neo-expressing NIH-3T3 cells. This decrease was concomitant with an increase in the mRNA concentration of the endogenous c-myc gene. FTO-2B rat hepatoma cells also showed modifications in gene expression when the neo gene was introduced by stable transfection or infection. In these cells an increase in both P-enolpyruvate carboxykinase (PEPCK) and tyrosine aminotransferase (TAT) mRNA was observed. These results suggest that neo gene expression may induce changes in the cells, which should be considered when neo-selected cells are used to deliver specific genes in different therapy approaches and in embryo manipulation.

3T3 Cells↗
PubMed1988

Protective effect of exogenous fructose-1,6-diphosphate in cardiogenic shock.

The effect of intravenous fructose-1,6-diphosphate (FDP) infusion on haemodynamic and biochemical variables was studied in dogs after ligation of the anterior descending branch of the left coronary artery. In the control series cardiogenic shock was present in every case 4 h after ligation. In FDP treated animals 4 h after ligation there was no fall in cardiac output and the systolic blood pressure was restored to pre-ligation values. Levels of serum creatine kinase isoenzyme (CK-MB), a highly specific indicator of myocardial cell damage, rose in the shocked (no FDP given) group, but remained low in the FDP treated group, equalling the levels measured in sham operated (no ligation) dogs. Samples of myocardium were taken from infarcted and adjacent normal regions 4 h after ligation for biochemical analysis. CK-MB concentrations in the infarcted region did not change from normal levels with FDP infusion; in the infarcted region lactate concentration (mumol.g-1 wet weight) fell from 18.48 in the control group to 7.90 in the FDP treated group. ATP levels in the infarcted region remained the same as those in the adjacent normal region with FDP treatment. It is concluded that infusion of FDP improves myocardial performance and metabolism following acute myocardial ischaemia.

Animals↗