Effect of the sodium/potassium ratio on glyceraldehyde 3-phosphate dehydrogenase interaction with red cell vesicles.

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the 31P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K (1977) Biochim. Biophys. Acta 464, 82--92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation changed induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na4 + K+)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew (1970) J. Physiol. London 207, 393--402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by 31P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na+ + K+)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.     

Relation between red cell membrane (Na + K)-ATPase and band 3 protein

This study is designed to examine the participation of the major red cell membrane protein, band 3 protein, in the chain which transmits information from the cardiac glycoside site on the external face of the cell (Na⁺ + K⁺)-ATPase to the megadalton glycolytic enzyme complex within the cell. The experiments show that the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, affects the resonance of 2,3-diphosphoglycerate, as does the cardiac glycoside cation transport inhibitor, ouabain. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor which indicates that there is coupling in the red cell between the (Na⁺ + K⁺)-ATPase and band 3 protein. Band 3 protein was separated from the membrane and partially purified following the technique of Yu and Steck ((1975) J. Biol. Chem. 250, 9170–9175). When glyceraldehyde-3-phosphate dehydrogenase was added to the separated band 3 protein preparation, addition of cardiac glycosides caused shifts in the ³¹P resonance of glyceraldehyde 3-phosphate. These experiments indicate that there is coupling between the (Na⁺ + K⁺)-ATPase and band 3 protein in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell.     

Membrane mediated link between ion transport and metabolism in human red cells

When 10^?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the ³¹P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na⁺ + K⁺)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na⁺ + K⁺)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10^?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na⁺ + K⁺)-ATPase and that the interaction is inhibited by 10^?6 M ouabain.     

Kinetic evidence for interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase.

The possibility of interaction between purified rabbit muscle aldolase and D-glyceraldehyde-3-phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system. The Km of the intermediary product, glyceraldehyde 3-phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde-3-phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) form of glyceraldehyde 3-phosphate, although in the given conditions the aldehyde leads to diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. We suggest that above a certain concentration of the enzymes the glyceraldehyde 3-phosphate produced by aldolase gets direct access to glyceraldehyde-3-phosphate dehydrogenase without participating in the aldehyde leads to diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.     

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

Isolation and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes.

A rapid and convenient procedure for isolating human glyceraldehyde-3-phosphate dehydrogenase from erythrocytes has been developed and yields enzyme with a specific activity of 33–52. The physical and catalytic properties of the enzyme are similar to those of rabbit muscle enzyme. Reassociation of freshly isolated human glyceraldehyde-3-phosphate dehydrogenase with washed erythrocyte membranes increases the specific activity and stability of the enzyme suggesting that enzyme-membrane interactions may have an important effect on the conformation and catalytic activity. That the human enzyme behaves as a dimer of dimers, similar to the behavior or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, is suggested by its half-of-the-sites reactivity toward 4-iodoacetamido-1-naphthol. The human enzyme binds nicotinamide hypoxanthine dinucleotide, a structural analog of NAD⁺, with negative cooperativity, further indicating its similarity to rabbit muscle enzyme.     

Isolation and characterization of an alkali metal ion-sensitive NAD+-dependent glyceraldehyde 3-phosphate dehydrogenase from germinating green gram (Phaseolus aurieus).

An alkali metal ion-sensitive NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase has been purified 250-fold from germinating green gram (Phaseolus aurieus). The purified enzyme shows a single protein band on gel electrophoresis. It has been shown to be a tetrameric protein (molecular weight 160,000) made up of apparently identical monomers (subunit molecular weight 40,000). It shows an $\text{A}{}_{280}\text{A}{}_{260}$ ratio equal to 1.38, which is not changed on treatment with animal charcoal or cellulosic ion exchangers. Direct estimation shows less than 0.07 mol bound NAD⁺/mol enzyme. Green gram glyceraldehyde 3-phosphate dehydrogenase is inhibited fairly strongly at physiological concentrations of Na⁺ ions. The inhibition is stronger at higher pH and lower protein concentration. Deproteinated extract, cysteine, and reduced glutathione reverse the Na⁺ ion inhibition. The effect of deproteinated extract is attributable to the presence of some SH-containing compounds. Potassium and rubidium ions have a mild activating effect at lower concentration (below 100 mm) and are inhibitory at higher, nonphysiological, concentrations. Ammonium and lithium ions have no effect. The inhibition due to Na⁺ ions is noncompetitive with respect to NAD⁺ and phosphate ions but competitive with respect to glyceraldehyde 3-phosphate, with K_i about 60 mm. Sodium ions protect the enzyme against proteolysis with trypsin. It is suggested that Na⁺ ions and the small molecular weight SH-compounds may possibly be involved in regulation of the overall rate of glycolysis via modulation of glyceraldehyde 3-phosphate dehydrogenase activity.     

Selective Inhibition by Actinomycin D of the Synthesis in Photosynthetic and Non-photosynthetic Enzymes During the Greening of Etiolated Bean Leaves

The effect of actinomycin D on the synthesis of the photosynthetic apparatus during illumination of etiolated leaves of Phaseolus vulgaris was studied. The increase of chlorophyll content and of the activities of some photosynthetic enzymes (NADPH diaphorase, ferredoxin, NADP⁺ glyceraldehyde-3-phosphate dehydrogenase) was compared with simultaneous measurements of the level of other enzymes not considered associated with photosynthesis (ornithine transcarbamylase, glucose-6-phosphate dehydrogenase, NAD⁺ glyceraldehyde-3-phosphate dehydrogenase).     

Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from the extremely halophilic archaebacterium Haloarcula vallismortis has been purified in a four step procedure to electrophoretic homogeneity. The enzyme is a tetramer with a relative molecular mass of 160000. It is strictly NAD⁺-dependent and exhibits its highest activity in 2 mol/l KCl at 45°C. Amino acid analysis and isoelectric focusing indicate an excess of acidic amino acids. Two parts of the primary sequence are reported. These peptides have been compared with glyceraldehyde 3-phosphate dehydrogenases from other archaebacteria, eubacteria and eucaryotes. The peptides show a high grade of similarity to glyceraldehyde 3-phosphate dehydrogenase from eucaryotes.Abbreviations BCA bicinchoninic acid - CTAB cetyltrimethyl ammonium bromide - DTE dithioerythritol - DTT dithiothreitol - GAP glyccraldehyde 3-phosphate - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

Cyclic AMP inhibition of reactions of glyceraldehyde 3-phosphate dehydrogenase

Adenosine 3′,5′-monophosphate (cyclic AMP) is an inhibitor of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate and benzaldehyde. Inhibition appears to be competitive toward glyceraldehyde 3-phosphate and of a mixed type toward NAD⁺. In the absence of arsenate a plot of $\text{1}\text{V}$ vs (I) is sigmoidal at constant concentrations of glyceraldehyde 3-phosphate and NAD⁺ and linear at constant concentrations of benzaldehyde and NAD⁺. Thus, sigmoidal inhibition plots are dependent on the nature of the aldehyde substrate as was found previously to be the case with inhibition of these reactions by highly branched acyl phosphates. In the presence of 0.013 m arsenate the plots of $\text{1}\text{V}$ vs [I] are linear.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

Proliferative and nutritional dependent regulation of glyceraldehyde-3-phosphate dehydrogenase expression in the rat liver

Glyceraldehyde-3-phosphate dehydrogenase is a multifunctional protein possessing numerous cytoplasmic and nuclear functions associated with cellular proliferation. Despite the emerging role of glyceraldehyde-3-phosphate dehydrogenase in regulating the proliferative process, there is a paucity of data regarding its expression and intracellular distribution in non-malignant proliferating hepatocytes. Thus the aim of the present study was to document the intracellular distribution of glyceraldehyde-3-phosphate dehydrogenase protein in proliferating hepatocytes derived from regenerating rat livers, and glyceraldehyde-3-phosphate dehydrogenase gene expression in fasted and re-fed rats following partial hepatectomy (PHx). Glyceraldehyde-3-phosphate dehydrogenase mRNA and protein expression were documented by Northern and Western blot analyses, respectively, at various times following 70% PHx in adult Sprague-Dawley rats. At 24 h post-surgery, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was significantly increased in both PHx and sham operated rats (P < 0.001), respectively. Despite the increase in glyceraldehyde-3-phosphate dehydrogenase mRNA expression in both groups, only PHx rats had a significant increase in the nuclear fraction of glyceraldehyde-3-phosphate dehydrogenase protein (threefold increase compared to sham and baseline levels, P < 0.01), cytoplasmic levels of glyceraldehyde-3-phosphate dehydrogenase protein remained unaltered in both groups. In terms of the effects of feeding and fasting on rats there were no significant differences in glyceraldehyde-3-phosphate dehydrogenase mRNA levels, whether fasted or refed, in rats that had undergone PHx, 8 h earlier. On the other hand, glyceraldehyde-3-phosphate dehydrogenase mRNA levels were significantly increased in refed compared to fasted sham operated rats 8 h following surgery. Serum insulin concentrations were higher in the refed PHx and sham groups compared to their fasted counterparts. The results of this study indicate that although glyceraldehyde-3-phosphate dehydrogenase mRNA are altered to the same extent in PHx and sham-operated rats following surgery, increases in the nuclear fraction of glyceraldehyde-3-phosphate dehydrogenase protein only occur in PHx rats. The results also indicate that glyceraldehyde-3-phosphate dehydrogenase expression is affected by the nutritional status of animals undergoing abdominal sham surgery.     

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD⁺-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD⁺–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD⁺ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N⁶-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD⁺ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD⁺); 80 (lactate+NAD⁺); 95 (lactate+semicarbazide+NAD⁺); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.     

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

     

A reappraisal of 31P NMR studies indicating enzyme complexation in red blood cells.

³¹P NMR and column fractionation studies do not substantiate the existence in solution of a complex of phosphoglycerate kinase and phosphoglycerate mutase or of one involving these enzymes and glyceraldehyde-3-phosphate dehydrogenase. It is shown that the small shifts $\text{(}\text{<}\text{3.2Hz)}$ in the ³¹P resonances of 2,3-bisphosphoglycerate which were interpreted to indicate the existence of such complexes (Fossel and Solomon (1977) BBA $\text{464}\text{, 82–92)}$ probably result from very small variations in pH (<0.1 unit). Further, no significant resonance shifts are detected in the presence of ouabain in glucose-depleted human red blood cells. An error analysis of the NMR data indicates that previously reported ouabain-induced shifts are within the noise level of the measurement and do not indicate the presence of enzyme complexes in the red cell.     

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Effects of extracellular potassium (K⁺) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whitten's medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K⁺ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K⁺-free medium. Millimole concentrations of K⁺ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K⁺ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K⁺ was higher than in media with 12 and 16 mM K⁺. These results suggest that extracellular K⁺ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K⁺ in maturation media impair cytoplasmic maturation.     

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.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

The connection between ionophore-mediated Ca-movements and intermediary metabolism in human red cells. II. Site and mode of glycolytic activation during Ca-loading

Human red cells (RBC) respond to moderate Ca²⁺-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca²⁺-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase (GAPDH/PGK)-steps. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca²⁺-extrusion rate and not to the loaded Ca²⁺. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca²⁺-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the GAPDH/PGK equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. NAD/NADH ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.