Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,in Human Cancer Cells: METABOLIC BASIS AND POTENTIAL CLINICAL IMPLICATIONS*

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD⁺, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD⁺ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.     

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells

NAD⁺ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD⁺ or NAD⁺ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD⁺ precursors for NAD⁺ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD⁺ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.     

Partial purification and kinetic characterization of transaldolase fromDictyostelium discoideum

Transaldolase was purified 42-fold fromDictyostelium discoideum and the resulting preparation exhibited stoichiometry. Kinetic analyses consisted of initial velocity and product inhibition studies in both the forward and the reverse directions. The enzyme exhibited ping-pong kinetics with sedoheptulose 7-phosphate adding first and erythrose 4-phosphate releasing first. TheK_m values for sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, and fructose 6-phosphate were 0.46, 0.072, 0.10, and 1.6 mM, respectively. TheK_i values for sedoheptulose 7-phosphate and erythrose 4-phosphate were 3.6 and 0.062 mM, respectively. Inorganic phosphate inhibited enzymatic activity and showed mixed-type inhibition when fructose 6-phosphate was varied. AK_i value of 35.2 mM was determined for inorganic phosphate.     

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116R^FK866. In this study, we investigated gene mutations in parental HCT116 and HCT116R^FK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD⁺ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116R^FK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.     

Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.     

Enzymes involved in the formation of glycerol 3-phosphate and the by-products dihydroxyacetone and glycerol in Zymomonas mobilis

Abstract In Zymomonas mobilis a novel pathway for the formation of glycerol 3-phosphate was identified by enzymatic studies and nuclear magnetic resonance spectroscopy. This pathway branches off from the Entner-Doudoroff pathway at the intermediate glyceraldehyde 3-phosphate and proceedes via dihydroxyacetone phosphate, dihydroxyacetone, glycerol to glycerol 3-phosphate. The reaction sequence is catalyzed by the enzymes triosephosphate isomerase (0.4 U (mg protein)⁻¹), dihydroxyacetone phosphatase (0.31 U (mg protein)⁻¹), dihydroxyacetone reductase (0.25 U (mg protein)⁻¹), and glycerokinase (0.08 mU (mg protein)⁻¹), respectively. The action of a postulated aldolase catalyzing the cleavage of fructose 6-phosphate to dihydroxyacetone and glyceraldehyde 3-phosphate could be excluded.     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.     

Inhibition of the glycolytic pathway by methylglyoxal in human platelets

The incubation of human platelets with methylglyoxal and glucose produces a rapid transformation of the ketoaldehyde to D-lactate by the glyoxalase system and a partial reduction in GSH. Glucose utilization is affected at the level of the glycolytic pathway. No effect of the ketoaldehyde on glycogenolysis and glucose oxidation through the hexose monophosphate shunt was demonstrated. Phosphofructokinase, fructose 1,6 diphosphate (F1, 6DP) aldolase, glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate mutase were mostly inhibited by methylglyoxal. A decrease in lactate and pyruvate formation and an accumulation of some glycolytic intermediates (fructose 1,6 diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate) was observed. Moreover methylglyoxal induced a fall in the metabolic ATP concentration. Since methylglyoxal is an intermediate of the glycolytic bypass system from dihydroxyacetone phosphate to D-lactate, it may be assumed that ketoaldehyde exerts a regulating effect on triose metabolism.     

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD⁺) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD⁺ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.Subject terms: Drug development, Cancer metabolism

     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Characterization of mammalian sedoheptulokinase and mechanism of formation of erythritol in sedoheptulokinase deficiency

Our aim was to identify the product formed by sedoheptulokinase and to understand the mechanism of formation of erythritol in patients with sedoheptulokinase deficiency. Mouse recombinant sedoheptulokinase was found to be virtually specific for sedoheptulose and its reaction product was identified as sedoheptulose 7-phosphate. Assays of sedoheptulose in plant extracts disclosed that this sugar is present in carrots ( approximately 7mumol/g) and in several fruits. Sedoheptulose 1-phosphate is shown to be a substrate for aldolase B, which cleaves it to dihydroxyacetone-phosphate and erythrose. This suggests that, in patients deficient in sedoheptulose-7-kinase, sedoheptulose is phosphorylated by fructokinase to sedoheptulose 1-phosphate. Cleavage of the latter by aldolase B would lead to the formation of erythrose, which would then be reduced to erythritol.     

Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD⁺ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD⁺ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD⁺-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD⁺ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.     

Endogenous NAMPT dampens chemokine expression and apoptotic responses in stressed tubular cells

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease and identification of new therapeutic targets is needed. Nicotinamide phosphoribosyltransferase (NAMPT) is both an extracellular and intracellular protein. Circulating NAMPT is increased in diabetics and in chronic kidney disease patients. The role of NAMPT in renal cell biology is poorly understood. NAMPT mRNA and protein were increased in the kidneys of rats with streptozotocin-induced diabetes. Immunohistochemistry localized NAMPT to glomerular and tubular cells in diabetic rats. The inflammatory cytokine TNFα increased NAMPT mRNA, protein and NAD production in cultured kidney human tubular cells. Exogenous NAMPT increased the mRNA expression of chemokines MCP-1 and RANTES. The NAMPT enzymatic activity inhibitor FK866 prevented these effects. By contrast, FK866 boosted TNFα-induced expression of MCP-1 and RANTES mRNA and endogenous NAMPT targeting by siRNA also had a proinflammatory effect. Furthermore, FK866 promoted tubular cell apoptosis in an inflammatory milieu containing the cytokines TNFα/IFNγ. In an inflammatory environment FK866 promoted tubular cell expression of the lethal cytokine TRAIL. These data are consistent with a role of endogenous NAMPT activity as an adaptive, protective response to an inflammatory milieu that differs from the proinflammatory activity of exogenous NAMPT. Thus, disruption of endogenous NAMPT function in stressed cells promotes tubular cell death and chemokine expression. This information may be relevant for the design of novel therapeutic strategies in DN.     

Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases

We have cloned an open reading frame from the Escherichia coli K-12 chromosome that had been assumed earlier to be a transaldolase or a transaldolase-related protein, termed MipB. Here we show that instead a novel enzyme activity, fructose-6-phosphate aldolase, is encoded by this open reading frame, which is the first report of an enzyme that catalyzes an aldol cleavage of fructose 6-phosphate from any organism. We propose the name FSA (for fructose-six phosphate aldolase; gene name fsa). The recombinant protein was purified to apparent homogeneity by anion exchange and gel permeation chromatography with a yield of 40 mg of protein from 1 liter of culture. By using electrospray tandem mass spectroscopy, a molecular weight of 22,998 per subunit was determined. From gel filtration a size of 257,000 (+/- 20,000) was calculated. The enzyme most likely forms either a decamer or dodecamer of identical subunits. The purified enzyme displayed a V(max) of 7 units mg(-)1 of protein for fructose 6-phosphate cleavage (at 30 degrees C, pH 8.5 in 50 mm glycylglycine buffer). For the aldolization reaction a V(max) of 45 units mg(-)1 of protein was found; K(m) values for the substrates were 9 mm for fructose 6-phosphate, 35 mm for dihydroxyacetone, and 0.8 mm for glyceraldehyde 3-phosphate. FSA did not utilize fructose, fructose 1-phosphate, fructose 1,6-bisphosphate, or dihydroxyacetone phosphate. FSA is not inhibited by EDTA which points to a metal-independent mode of action. The lysine 85 residue is essential for its action as its exchange to arginine (K85R) resulted in complete loss of activity in line with the assumption that the reaction mechanism involves a Schiff base formation through this lysine residue (class I aldolase). Another fsa-related gene, talC of Escherichia coli, was shown to also encode fructose-6-phosphate aldolase activity and not a transaldolase as proposed earlier.     

Metabolomics Analysis of Metabolic Effects of Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibition on Human Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide–consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry–based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level.     

Biosynthesis of bacterial glycogen: characterization of adenosine diphosphate glucose synthetases from Enterobacter hafniae and Aeromonas hydrophila

Enterobacter hafniae and Aeromonas hydrophila ADPglucose synthetases were purified approximately 39-and 61-fold, respectively, over the crude extract. Both enzymes were heat stable at 60°C in the presence of inorganic phosphate. The molecular weights of both enzymes were approximately 200,000 which are similar to other enteric ADPglucose synthetases studied. Based on kinetic results obtained from the partially purified enzymes, the E. hafniae enzyme is activated twofold by phospho-enolpyruvate while the A. hydrophila enzyme is activated twofold by fructose 6-P and 1.5-fold by fructose 1,6 bis-phosphate. The E. hafniae enzyme activity is strongly inhibited by AMP and ADP and the inhibition can be partially reversed by P-enolpyruvate. ADP is the most effective inhibitor of the A. hydrophila enzyme and its inhibiton can be partially overcome by the presence of the activators fructose 6-P and fructose 1,6-P₂. These kinetic results show that the allosteric properties of the E. hafniae enzyme are distinctly different from the ADPglucose synthetases of those previously studied from bacteria of the genus Enterobacter. Although the A. hydrophila enzyme is activated by fructose 1,6-P₂, its allosteric properties are quite different than those observed for ADPglucose synthetase of the Enterobacteriaceae.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - glucose 1-P glucose 1-phosphate - Bicine N,N-bis(2 hydroxyethyl)glycine - fructose 6-P fructose 6-phosphate - Mes 2(N-morpholino)-ethane sulfonic acid - fructose 1,6-P₂ fructose 1,6 bis-phosphate - DTE dithioerythritol; pyridoxal-P, pyridoxal-phosphate - fructose 1-P fructose 1-phosphate - P-enolpyruvate phospho-enolpyruvate - 1,6 hexanediol bis-P 1,6 hexanediol bis-phosphate; glucose 6-P, glucose 6-phosphate - dihydroxyacetone-P dihydroxyacetone phosphate - 1-glycerol-3-P 1-glycerol-3-phosphate - erythrose 4-P erythrose 4-phosphate - 2-P-glycerate 2-phosphoglycerate - sedoheptulose 1,7-P₂ sedoheptulose 1,7 bis-phosphate - 3-P-glycerate 3-phosphoglycerate - mannose-6-P mannose-6-phosphate     

Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles

Summary A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate d-fructose-6-phosphate to d-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into d-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the d-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-d-glycerate. Concomitantly the electrons are transported via NAD⁺, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Metabolic effects of D-glyceraldehyde in isolated hepatocytes.

The effects of D-glyceraldehyde on the hepatocyte contents of various metabolites were examined and compared with the effects of fructose, glycerol and dihydroxyacetone, which all enter the glycolytic/gluconeogenic pathways at the triose phosphate level. D-Glyceraldehyde (10 MM) caused a substantial depletion of hepatocyte ATP, as did equimolar concentrations of fructose and glycerol. D-Glyceraldehyde and fructose each caused a 2-fold increase in fructose 1,6-bisphosphate and the accumulation of millimolar quantities of fructose 1-phosphate in the cells. D-Glyceraldehyde caused an increase in the glycerol 3-phosphate content and a decrease in the dihydroxyacetone phosphate content, whereas dihydroxyacetone increased the content of both metabolites. The increase in the [glycerol 3-phosphate]/[dihydroxyacetone phosphate] ratio caused by D-glyceraldehyde was not accompanied by a change in the cytoplasmic [NAD+]/[NADH] ratio, as indicated by the unchanged [lactate]/[pyruvate] ratio. The accumulation of fructose 1-phosphate from D-glyceraldehyde and dihydroxyacetone phosphate in the hepatocyte can account for the depletion of the intracellular content of the latter. Presumably ATP is depleted as the result of the accumulation of millimolar amounts of a phosphorylated intermediate, as is the case with fructose and glycerol. It is suggested that the accumulation of fructose 1-phosphate during hepatic fructose metabolism is the result of a temporary increase in the D-glyceraldehyde concentration because of the high rate of fructose phosphorylation compared with triokinase activity. The equilibrium constant of aldolase favours the formation and thus the accumulation of fructose 1-phosphate.