Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications.

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.     

On the differential release of glycolytic enzymes from cellular structure

In an endeavour to extend the available information on the biological significance of the interactions between glycolytic enzymes and cellular ultrastructure, the role of release of enzymes from digitonized fibroblasts has been studied. Lactate dehydrogenase and phosphofructokinase were rapidly and quantitatively eluted under the experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase and aldolase were retained to an appreciably greater extent by the cells. This differential release of glycolytic enzymes has been related to the known binding propensities between those enzymes and subcellular structures, and are interpreted as providing additional confirmatory evidence of the importance of aldolase and glyceraldehyde-3-phosphate dehydrogenase, in particular, to these associations. The data also shed light on the order of binding of these glycolytic components - phosphofructokinase being indicated as binding subsequently (and probably separately) to aldolase and glyceraldehyde-3-phosphate dehydrogenase. These results have been discussed in relation to the available data on the associations between glycolytic enzymes and cellular structure, the possible physiological significance of this phenomenon, and the access to these problems provided by the present technique.     

A quantitative evaluation of the effect of enzyme complexes on the glycolytic rate in vivo: mathematical modeling of the glycolytic complex

The cellular distribution of free and bound glycolytic enzymes in vivo was estimated by means of a model based on previously determined association constants for individual binding interactions and in vivo protein concentrations. The calculations revealed that a significant proportion of the enzymes would be either associated with F-actin, or bound in binary enzyme-enzyme complexes in vivo. An analysis of the relative concentration, and relative activity, of F-actin-bound enzymes suggested that a complete glycolytic complex, composed of all enzymatic steps from phosphofructokinase (PFK) to lactate dehydrogenase (LDH) does not exist. This was indicated by a very low concentration of F-actin-associated phosphoglycerate kinase (PGK) and by a very low activity of F-actin bound aldolase and PGK; this model showed that aldolase and PGK would be absent from any F-actin bound complex. An analysis of soluble enzyme-enzyme associations indicated that formation of binary enzyme complexes may lead to an increased overall flux through glyceraldehyde 3-phosphate dehydrogenase and LDH, but would serve to decrease flux through PFK and aldolase. A 1.4-fold activation of PFK, which occurs when the soluble enzyme binds to F-actin, suggested that reversible binding of PFK to F-actin may represent a novel cellular mechanism for controlling glycolytic flux during periods of increased metabolic demand by controlling the key regulatory enzyme of glycolysis.     

Antivibrionic activity of some glycolytic cycle enzymes of animal cells.

The antivibrionic activity of crystalline preparations of five enzymes of the glycolytic cycle of animals cells was investigated. Phosphorylase "a" (0.5 mg/ml), aldolase (15 mg/ml) and pyruvate kinase (0.1 mg/ml) were found to inhibit the proliferation of Vibrio cholerae cells; phosphoglucomutase and glyceraldehyde-3-phosphate dehydrogenase at a concentration of 0.25 mg/ml were found to be vibriocidal. A mixture of these enzymes containing 0.062 mg/ml of phosphorylase "a" and 0.125 mg/ml of each phosphoglucomutase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase showed vibriocidal activity.     

Ionic strength dependence of F-actin and glycolytic enzyme associations: a Brownian dynamics simulations approach

The association of glycolytic enzymes with F-actin is proposed to be one mechanism by which these enzymes are compartmentalized, and, as a result, may possibly play important roles for: regulation of the glycolytic pathway, potential substrate channeling, and increasing glycolytic flux. Historically, in vitro experiments have shown that many enzyme/actin interactions are dependent on ionic strength. Herein, Brownian dynamics (BD) examines how ionic strength impacts the energetics of the association of F-actin with the glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase (aldolase), and triose phosphate isomerase (TPI). The BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases but also suggest that these interactions are significant, at physiological ionic strengths. Furthermore, BD agrees with experiments that muscle LDH, aldolase, and GAPDH interact significantly with F-actin whereas TPI does not. BD indicates similarities in binding regions for aldolase and LDH among the different species investigated. Furthermore, the residues responsible for salt bridge formation in stable complexes persist as ionic strength increases. This suggests the importance of the residues determined for these binary complexes and specificity of the interactions. That these interactions are conserved across species, and there appears to be a general trend among the enzymes, support the importance of these enzyme-F-actin interactions in creating initial complexes critical for compartmentation.     

Glycolytic enzyme interactions with yeast and skeletal muscle F-actin

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that "actin binds enzymes", which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.     

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer.     

The effect of enzyme-enzyme complexes on the overall glycolytic rate in vivo.

Recent studies have demonstrated that most glycolytic enzymes can reversibly associate to form heterogeneous enzyme-enzyme (binary) complexes in vitro. However, kinetic analysis of these complexes has shown that the individual enzymes have a varied response to complex formation: some enzymes are inhibited, some are activated and some are unaffected. In order to determine the potential role of binary complexes in regulating glycolytic flux, we have mathematically calculated enzyme distributions and activities using data from in vitro binding and kinetic studies. These calculations suggest that, overall, formation of binary complexes would lower flux through phosphofructokinase and aldolase, would increase flux through glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and would not affect flux through triosephosphate isomerase, phosphoglycerate kinase and pyruvate kinase. The implications of these results are discussed with respect to the effect of complex formation on overall glycolytic flux and on the flux through individual enzyme loci.     

Changes in enzyme binding and activity during aestivation in the frog Neobatrachus pelobatoides

1. The proportion of aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) associated with the particulate fraction of a cell was measured in aestivating and non-aestivating Neobatrachus pelobatoides. 2. Reduced binding of these enzymes was found in the brain, indicating lower glycolytic flux. This was not correlated to metabolic rate suggesting that glycolytic rate was reduced in this tissue in the early stages of aestivation, possibly due to a change in fuel use. 3. Measurement of total enzyme levels showed that the liver of aestivating frogs had less GAPDH and less aldolase than non-aestivating frogs.     

Enzymatic analysis of the pathways of glucose catabolism and gluconeogenesis in Pseudomonas citronellolis

Extracts of Pseudomonas citronellolis cells grown on glucose or gluconate possessed all the enzymes of the Entner-Doudoroff pathway. Gluconokinase and either or both 6-phosphogluconate dehydratase and KDPG aldolase were induced by growth on these substrates. Glucose and gluconate dehydrogenases and 6-phosphofructokinase were not detected. Thus catabolism of glucose proceeds via an inducible Entner-Doudoroff pathway. Metabolism of glyceraldehyde 3-phosphate apparently proceeded via glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. These same enzymes plus triose phosphate isomerase were present in lactate-grown cells indicating that synthesis of triose phosphates from gluconeogenic substrates also occurs via this pathway. Extracts of lactate grown-cells possessed fructose diphosphatase and phosphohexoisomerase but apparently lacked fructose diphosphate aldolase thus indicating either the presence of an aldolase with unusual properties or requirements or an alternative pathway for the conversion of triose phosphate to fructose disphosphate. Cells contained two species of glyceraldehyde 3-phosphate dehydrogenase, one an NAD-dependent enzyme which predominated when the organism was grown on glycolytic substrates and the other, an NADP-dependent enzyme which predominated when the organism was grown on gluconeogenic substrates.     

The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue

1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.     

The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase

The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.     

Three enzymes of carbon metabolism or their antigenic analogs in pea leaf nuclei.

Antigens closely resembling or identical to the three glycolytic enzyme proteins phosphate-glycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase are found in situ in the nucleus of the leaf mesophyll cells of pea (Pisum sativum L.). These proteins have already been identified in vertebrate nuclei. Apparently, these enzymes are nuclear proteins with "secondary" roles not directly related to their enzymatic function in carbon metabolism in both animals and plants.     

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.     

Substrate-induced dissociation of glycerol-3-phosphate dehydrogenase and its complex formation with fructose-bisphosphate aldolase

A threefold decrease in specific activity of glycerol-3-phosphate dehydrogenase was found on going from 800 nM to 10 nM enzyme concentration. According to ultracentrifugal analyses the dimeric glycerol-3-phosphate dehydrogenase (molecular weight 78,000) dissociates into monomers in the equilibrium mixture of its substrates and products. The concentration-dependent decrease in the specific activity is interpreted as a consequence of subunit dissociation and the estimated dissociation constants are 0.7 micro M and 3.5 micro M at 38 degrees C and 20 degrees C respectively. According to active-enzyme-band centrifugation experiments and kinetic analysis aldolase forms a complex with glycerol-3-phosphate dehydrogenase and this complex formation influences the specific activity of the dehydrogenase. The interaction between glycerol-3-phosphate dehydrogenase and aldolase can provide a regulatory mechanism at the branching point of glycolytic and lipid metabolic pathways.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

Biochemistry of Coxiella burnetii: Embden-Meyerhof pathway

Purified preparations of Coxiella burnetii were examined for enzymes of the glycolytic pathway. Glucose-phosphate isomerase, fructose-1,6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase were shown to be present in C. burnetii extracts. Heat-killed C. burnetii purified with normal yolk sacs demonstrated no activity after disruption. Aldolase was shown to be of the class II type by complete inhibition of activity in the presence of 8 x 10(-3)m ethylenediaminetetraacetic acid. The host enzyme activity (normal and infected yolk sacs) was not affected by the same treatment. When cellulose acetate electrophoresis was performed on the extracts, aldolase from both normal and infected yolk sacs exhibited five isozyme bands, whereas aldolase from the C. burnetii extract appeared as a single band.