Binding Of Glycolytic Enzymes Research Articles

Hemoglobin S Polymerization Can be Inhibited By Alterations in Erythrocyte Glycolysis

Sickle cell disease (SCD) is caused by the polymerization of deoxy Hemoglobin S (HbS) resulting in lifelong complications including anemia, infections, stroke, tissue damage, organ failure, intense painful episodes, and premature death. Each year more than 300,000 children are born with SCD, and greater than 95% of these children live in resource-poor countries. Although bone marrow transplantation and/or gene therapy are curative or potentially curative treatments for SCD, these options are not available for the vast majority of people with SCD. The most effective palliative treatment for SCD is hydroxyurea, a drug that was approved by the FDA in 1998. Clearly there is a global need for low cost, portable treatments for SCD. We have previously shown that erythrocytes sense oxygen through reversible binding of deoxy Hb to the cytoplasmic domain of Band 3 using 3 lines of transgenic mice: 1) a mouse in which the mouse Hb/glycolytic enzyme binding region of Band 3 is replaced with the equivalent human sequences (humanized), 2) a humanized mouse lacking the Band 3 Hb binding site (-Hb) and 3) a humanized mouse with a deletion of glycolytic enzyme binding sites, resulting in increased affinity for deoxy Hb (HI affinity). Complete blood counts of these animals demonstrated only a mild increase in the MCV for the -Hb mice (p<0.03) compared to the humanized control. In the humanized control mouse, under oxygenated conditions glycolytic enzymes associate with Band 3, inhibiting glycolysis. In deoxy conditions, deoxy Hb displaces the glycolytic enzymes, activating glycolysis. In the -Hb mouse, the glycolytic enzymes were permanently bound to Band 3 (inactive glycolysis), while in the HI affinity mouse, the glycolytic enzymes were constitutively in the cytoplasm. (Chu et al. Blood 128:2708-16, 2016, Zhang et al. JBC 284:2519-25, 2019, Zhou et al. Sci Adv. 5:4466, 2019). To determine whether the alteration in glycolysis had effects on the polymerization of HbS, each of the mutant mouse strains were crossed to the Townes SCD mouse model. The end result was 3 different strains of animals that were 1) homozygous for one of the Band 3 mutations, 2) homozygous for the human alpha globin genes and 3) either HbA/HbA, HbA/HbS or HbS/HbS. Changes in CBC were related to the beta globin genotype. An elevated MCV was observed in the -Hb animals of all 3 genotypes (p<0.02). We compared the rate of sickling of S/S cells (Li et al. PNAS 114 (5) E689-E696, 2017) and showed that the -Hb S/S mice had a 16% increase in both the absolute number of sickled cells and the rate of appearance of the sickled cells relative to the humanized control (p<0.02). In contrast, sickling of HI affinity S/S cells was inhibited. We hypothesized that alterations in glycolysis in the -Hb and HI affinity erythrocytes were responsible for the changes in HbS polymerization. To test this hypothesis, we analyzed the metabolites in erythrocytes from our mutant mice, including those with HbA/A, HbA/S and HbS/S. We found that the ADP concentration was 2-fold higher in -Hb S/S cells compared to HI affinity S/S cells (p <0.05). The relative levels of ATP were 1.5 fold lower in -Hb S/S cells compared to HI affinity S/S cells (p <0.05). Pentose phosphate pathway (PPP) intermediates were elevated in HI Affinity S/S cells compared to -Hb S/S cells, indicating that the Hi Affinity S/S mice have recovered PPP activity. To test whether altering glycolysis chemically could alter sickling, we treated humanized S/S cells with phosphoenolpyruvate (PEP), a membrane-permeable, rate-limiting glycolytic substrate that is rapidly metabolized to lactate and/or processed up the glycolysis pathway to generate 2,3-bisphosphoglycerate, which can initiate the PPP. We found that PEP treatment led to a dose dependent suppression of sickling compared to untreated cells (p<10-16). At a PEP concentration of 10mM, sickling was reduced by 15% compared to untreated cells with no effects on either membrane stability or MCV (p<10-15). In summary, we have shown that alterations in glycolysis can enhance or suppresses the sickling of red blood cells in a mouse model. Our results offer the prospect of a non-invasive, dietary therapy to potentially inhibit sickling and improve the outcome for SCD patients in resource poor areas of the world. Disclosures Low: Umoja BioPharma Inc: Other: Stockholder, Founder; Novosteo Inc: Other: Stockholder, Founder; Erythrocure Inc.: Other: Stockholder, Founder; On Target Laboratories Inc: Other: Stockholder, Founder; HuLow LLC: Other: Stockholder, Founder.

AltitudeOmics: Red Blood Cell Metabolic Adaptation to High Altitude Hypoxia.

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia through the so-called oxygen-dependent metabolic regulation, which involves the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 3. This mechanism promotes the accumulation of 2,3-DPG, stabilizing the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the Bohr effect. Despite in vitro studies, in vivo adaptations to hypoxia have not yet been completely elucidated. Within the framework of the AltitudeOmics study, erythrocytes were collected from 21 healthy volunteers at sea level, after exposure to high altitude (5260 m) for 1, 7, and 16 days, and following reascent after 7 days at 1525 m. UHPLC-MS metabolomics results were correlated to physiological and athletic performance parameters. Immediate metabolic adaptations were noted as early as a few hours from ascending to >5000 m, and maintained for 16 days at high altitude. Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide, and sulfur/H2S metabolism. Metabolic adaptations were preserved 1 week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Deoxygenation of leucofiltered erythrocyte concentrates preserves proteome stability during storage in the blood bank.

While prospective clinical trials are currently underway in several countries throughout the world1, the safety and effectiveness of long-stored erythrocyte concentrates have been questioned by controversial retrospective clinical evidence2,3 and accumulating biochemical investigations4–6. Laboratory investigations have recently highlighted that a significant number of biochemical and mechanical lesions accumulate in erythrocyte concentrates during their storage in the blood bank. These alterations, often referred to as “storage lesions”, include: (i) the progressive impairment of cell metabolism, leading to the consumption of high energy phosphate compounds (ATP and 2,3-diphosphoglycerate)7; (ii) the accumulation of oxidative stress, resulting in the oxidation8, non-enzymatic glycosylation9, fragmentation or aggregation of the red blood cell (RBC) membrane10–13 and cytosolic proteins14; (iii) the progressive loss of metabolic modulation15, as a result of oxidative stress (especially in non-leucoreduced units)16 and protease activity-dependent fragmentation of the N-terminal cytosolic domain of band 317, which results in impairment of the regulatory function of the so-called “respiratory metabolon”18,19; (iv) the progressive oxidation of lipids and deregulation of lipid homeostasis20,21; (v) the exacerbation of membrane blebbing and shedding of microvesicles, a self-protective mechanism22, closely mimicking apoptosis23, which allows cells to get rid of no-longer functional or irreversibly altered proteins and lipids24, while retaining rheological properties of the donor25, which might result in pro-immunogenic potential in the recipient, together with newly appearing storage-dependent biomarkers promoting RBC phagocytosis, such as phosphatidylserine exposure; and (vi) microvesiculation events that compromise RBC morphology (changes from a discocytic to a spheroechnicocytic or spherocytic phenotype), which ends up impairing the surface-to-volume ratio, thereby increasing osmotic fragility26,27, and increasing membrane rigidity, as a result of progressive leaching and intercalation of the plasticizers (such as DEHP) into the membrane lipid bilayer28. In the light of such changes, alternative storage strategies have been proposed over the years, such as deoxygenation of packed erythrocyte concentrates through other methods29,30. The rationale underpinning such a strategy is that deoxygenation of erythrocyte concentrates would remove the main substrate for the production of reactive oxygen species, while promoting energy metabolism through the Embden-Meyerhoff pathway in the light of the oxygen-dependent metabolic modulation mediated by the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 318,19. Early studies by independent groups (and through independent processing strategies29,30) found deoxygenation-dependent improvement of hemolysis and morphological parameters, as well as prolonged preservation of high energy phosphate compounds29–31, though at the expenses of the NADPH-generating potential via the pentose phosphate pathway, as gleaned through metabolomics approaches31. In the light of these encouraging results, we decided to apply a proteomics workflow to understand whether the RBC membrane proteome of erythrocytes stored under deoxygenation in leucofiltered-units is better preserved than in untreated controls. In our previous investigation on deoxygenated units of non-leucofiltered erythrocyte concentrates12 we concluded that the overall spot numbers in two-dimensional electrophoresis (2DE) gels could represent a diagnostic marker of proteome homeostasis during storage in the blood bank. Indeed, RBC are devoid of nuclei and organelles and are, therefore, incapable of synthesising proteins de novo. Any alteration to the overall number of spots in 2DE gels can, therefore, be attributed to storage-dependent effects on the membrane proteome, which we previously identified as fragmentation and aggregation phenomena during the first two-three weeks of storage (increased number of detected spots), followed by vesiculation of damaged proteins (decreased number of spots)11,12. Furthermore, in control units, we previously correlated the storage-dependent alteration of the erythrocyte membrane proteome stability to an impairment in osmotic fragility26. Concordantly, in the present study we performed 2DE assays of the membrane proteome of leucofiltered erythrocytes stored under control conditions or in a deoxygenated environment (following oxygen removal, as previously described)30. As a result, we could confirm that deoxygenation had beneficial effects in terms of membrane proteome homeostasis and preserved osmotic resistance.

Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes

Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.

Mapping of the Glycolytic Enzyme Binding Sites on Human Erythrocyte Membrane Band 3.

Glycolytic enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.

Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC 50 of 88.6 ± 5.3 μM and a t 0.5 of 89.7 ± 7.2 min, with 50 μM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 μM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 μM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4 ± 3.6% and 41.0 ± 15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Characterization of Glycolytic Enzyme Complexes on Murine Erythrocyte Membranes.

Glycolytic enzymes have been recently shown to exist as multi-enzyme complexes in association with the cytoplasmic domain of band 3 at the inner surface of the human erythrocyte membrane. Because several of the glycolytic enzyme binding sites have been mapped to sequences near the NH2-terminus of band 3 (DDYED and EEYED) that are not conserved in mice (EEVLE and EELEN), the question naturally arose whether the existence of glycolytic enzyme complexes on erythrocyte membranes might be only a product of recent evolution. To test this hypothesis, fresh murine erythrocytes were fixed and stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and carbonic anhydrase II (CA II was used as a control, since it binds to a distant site near the COOH-terminus of band 3). Importantly, analysis of intact murine erythrocytes by confocal microscopy demonstrated that all of the above enzymes are localized to the membrane in oxygenated cells. In contrast, upon deoxygenation of the intact cells, release of the glycolytic enzymes (but not CA II) from the erythrocyte membrane and their uniform redistribution throughout the cytoplasm is observed. Because deoxyhemoglobin has been shown in human erythrocytes to compete with glycolytic enzymes (but not with CA II) for a common binding site at the NH2-terminus of band 3, these data argue that murine band 3, despite its weak homology to human band 3, still constitutes an organization center for glycolytic enzymes on the erythrocyte membrane. To further test this hypothesis, erythrocytes from band 3 knockout mice were similarly examined by confocal microscopy. Not surprisingly, all of the enzymes in all of the cells were evenly distributed throughout the cytoplasm, regardless of the oxygenation state of the cell. Further, immunoblot analyses demonstrated that glycolytic enzyme content of the band 3 knockout erythrocytes was measurably reduced compared to healthy mice, suggesting that the anion transporter may also contribute to enzyme stabilization during the lifetime of the erythrocyte. Finally, to determine whether the integrity of other membrane structures might impact the assembly of glycolytic enzyme complexes on the erythrocyte membrane, α-spectrin deficient mice were also examined for their enzyme distributions. Curiously, > 50% of the cells in any field exhibited glycolytic enzyme staining throughout the cytoplasm, with the remainder showing mainly membrane staining. Conceivably, the stabiity of glycolytic enzyme complexes on the membrane may also depend on the integrity of the membrane skeleton. Taken together, these data argue that glycolytic enzymes assemble in an oxygenation-dependent manner into complexes on murine erythrocyte membranes and that the stability of these complexes depends on the presence of band 3 and to a lesser extent α-spectrin. Supported by NIH grant GM24417.

Binding of glycolytic enzymes in adductor muscle of Iceland scallop Chlamys islandica is altered by reproductive status

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 268:141-149 (2004) - doi:10.3354/meps268141 Binding of glycolytic enzymes in adductor muscle of Iceland scallop Chlamys islandica is altered by reproductive status Katherina Brokordt1,2, Helga Guderley1,* 1Département de Biologie and Quebec-Océan (Le Groupe Interinstitutionnel de Recherches Océanographique du Québec), Université Laval, Quebec City, Quebec G1K 7P4, Canada 2Present address: Departamento de Ecología, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile *Corresponding author. Email: helga.guderley@bio.ulaval.ca ABSTRACT: During gonad growth and spawning by the Iceland scallop Chlamys islandica, muscle glycogen levels decrease in parallel with the activities of several glycolytic enzymes, including glycogen phosphorylase (GP), pyruvate kinase (PK) and octopine dehydrogenase (ODH), even though muscle protein concentrations remain unchanged. These parallel changes suggested that binding of glycolytic enzymes to glycogen influences their activities. Decreases in the amount of glycogen in the muscle would reduce the number of binding sites for these enzymes and decrease measurable activities or accelerate their degradation. We tested this hypothesis by evaluating the impact of reproductive status on the activity and intracellular distribution of GP, PK and ODH in adductor muscle of inactive Iceland scallops. Glycogen, but not protein, levels decreased markedly during gonad maturation and spawning. GP and ODH activities decreased in parallel with glycogen, while PK activity did not change. GP showed the highest binding both to glycogen (9 to 19.5% of activity bound) and to the particulate fraction (37 to 53%). As the units of GP bound per mg glycogen did not change with maturation and spawning, the total units of GP bound to glycogen decreased with maturation and spawning in females and males. For the enzymes measured, more activity was bound to the particulate fraction than to glycogen. The units of ODH bound to glycogen and to the particulate fraction decreased during maturation and spawning in females. Within a reproductive state and within a cellular fraction, the relative activities of the glycolytic enzymes (ODH/GP and PK/GP) were quite similar, suggesting conservation of the stoichiometry of glycolytic enzyme capacities in a given cellular fraction. Enzyme-binding to the particulate fraction of scallop adductor muscle shifts with long-term physiological change. Our results are compatible with the interpretation that the intracellular localization of enzymes influences their rates of synthesis and degradation. KEY WORDS: Enzyme-binding · Glycogen · Glycogen particles · Glycolytic enzymes · Scallops · Reproduction · Reproductive cost · Chlamys islandica Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 268. Online publication date: March 09, 2004 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2004 Inter-Research.

Detachment of glycolytic enzymes from cytoskeleton of Lewis lung carcinoma and colon adenocarcinoma cells induced by clotrimazole and its correlation to cell viability and morphology

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We report here that clotrimazole (l-(α-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, induced a dose-dependent detachment of the glycolytic enzymes, phosphofructokinase (ATP: d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase ( d-fructose-l,6-bisphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton membrane, and would also affect cytoskeleton structure and cell shape. We show here that clotrimazole decreased the viability of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. After 3 h of incubation with clotrimazole, complete cell destruction was detected. Ultrastructural cell damage was manifested by disintegration of the outer membrane by scanning electron microscopy (SEM). The detachment of glycolytic enzymes from cytoskeleton, induced by clotrimazole, preceded the decrease in cell viability, which indicates that this is an early effect and not a result of cell death. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation, and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by clotrimazole, as well as its reported inhibitory action on cell proliferation, makes this drug the most promising agent in the treatment of cancer.

Reversible binding of glycolytic enzymes and size change in the actin-containing filaments of the frog skeletal muscle.

Electron microscopy was used in order to study the change of the actin-containing filaments when the bound glycolytic enzymes were removed by a high ionic strength solution. The effectiveness of the extraction medium was checked by estimating the aldolase activity released. Evidence was provided that the larger diameter of the actin filaments in the I-bands in comparison with the A-bands can be accounted for by the bound glycolytic enzymes.

Detachment of the Glycolytic Enzymes, Phosphofructokinase and Aldolase, from Cytoskeleton of Melanoma Cells, Induced by Local Anesthetics

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. An important mechanism that controls glycolysis is the reversible binding of glycolytic enzymes to cytoskeleton. We report here that the local anesthetics, lidocaine and bupivacaine, induced a dose-dependent detachment of the glycolytic enzymes, phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13), from cytoskeleton of B16 melanoma cells. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of cytoskeleton-membrane and would also affect cytoskeleton structure. We show here that the local anesthetics decreased the viability of melanoma cells. The detachment of the glycolytic enzymes from cytoskeleton, induced by the drugs, preceded melanoma cell death, which indicates that this is an early effect and not a result of cell death. Bupivacaine was more potent than lidocaine both on the glycolytic enzymes and on cell viability. The present results suggest that local anesthetics, and especially bupivacaine, are promising drugs for the treatment of melanoma.

Taxol (paclitaxel) induces a detachment of phosphofructokinase from cytoskeleton of melanoma cells and decreases the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP

Glucose utilization through glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Here we report of a novel mechanism of action of taxol (paclitaxel; Baccatin III N-benzyl-β-phenylisoserine ester), the anti-microtubule agent with remarkable anticancer activity. We show that taxol affects both levels of regulation of glycolysis in melanoma cells; it decreases the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis, and also causes a detachment of phosphofructokinase (ATP: d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis, from the cytoskeleton of B16 melanoma cells. These effects of taxol were dose-dependent, and preceded the decrease in ATP levels and cell viability. Thus, taxol not only inhibits the essential dynamic processes of microtubule network, but also reduces glycolysis, through the novel mechanisms described here.

Insulin Stimulates Binding of Phosphofructokinase to Cytoskeleton and Increases Glucose 1,6-Bisphosphate Levels in NIH-3T3 Fibroblasts, Which Is Prevented by Calmodulin Antagonists

We report here a novel mechanism of insulin action in cultures of NIH-3T3 fibroblasts. Our experiments revealed that in these cells, insulin induced a rapid and transient increase in cytoskeleton-bound phosphofructokinase (EC 2.7.1.11), the rate-limiting enzyme in glycolysis, with a corresponding decrease in soluble (cytosolic) activity. Insulin also induced a slower increase in the levels of glucose 1,6-bisphosphate, the potent activator of cytosolic glycolysis. Both the rapid and the slower stimulatory actions of insulin were prevented by treatment with structurally different calmodulin antagonists, which strongly suggest that calmodulin is involved in these effects of insulin. The present and our previous experiments in muscle suggest that rapid, Ca2+-calmodulin-mediated increase in the binding of glycolytic enzymes to cytoskeleton, as well as the slower increase in glucose 1,6-bisphosphate, may be a general mechanism, in different cells, in signal transduction of insulin.

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues.

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 mumol/g wet weight and in muscle led to its increase from 3.64 to 25.1 mumol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Calmodulin Antagonists and Cell Energy Metabolism in Health and Disease

zymes are known to be regulated by reversible bindCalmodulin is a multifunctional Ca-binding proing to the cytoskeleton (8, for reviews, see 1,9,10). tein that has been implicated in the regulation of They bind both to actin filaments and to tubulin/ numerous cellular events. The Ca-calmodulin commicrotubules (8–15). The binding of glycolytic enplex plays a central role in the control of cell energy zymes to cytoskeleton was demonstrated both in metabolism (for previous review, see 1). It has a dual vitro, using purified components (16–23), and in effect on cell ATP-producing systems; while an invivo, in whole tissues (7,24–26), as well as in differcrease in intracellular Ca at physiological range ent cultured cells (27–29). Glycolytic enzymes in of concentrations exerts (through Ca-calmodulin their cytoskeleton-bound form are known to be more complex) stimulatory effects on cell energy metaboactive than their soluble form (9). Their activation, lism, Ca at high pathological concentration causes as well as their proximity to each other when bound a decrease in cell ATP and cell damage, characteristo the cytoskeleton, provides a more efficient glycotic of various diseases. lytic flux. Cytoskeletal glycolysis provides local ATP The ATP-producing systems in the different cell in the vicinity of the cytoskeleton, which is known compartments, which are controlled by the Ca-calto interact dynamically with the plasma membrane modulin complex, are: (a) cytosolic glycolysis, (b) cyupon membrane-induced events. Binding of glycotoskeletal glycolysis, and (c) mitochondrial oxidative lytic enzymes to cytoskeleton also affects cell strucmetabolism, which is linked to mitochondrial-bound ture, as glycolytic enzymes were found to cross-link hexokinase (HK). actin-containing filaments into ordered supramolecThe chief allosteric regulator of cytosolic glycolysis ular structures (9). Many factors and conditions conin extrahepatic tissues is glucose 1,6-bisphosphate trol the binding of glycolytic enzymes to the cytoskel(Glc-1,6-P2) (for reviews, see Refs. 1–5). This cometon (1). pound acts as an intracellular signal which, through Hexokinase is the only glycolytic enzyme which its allosteric effects on the key enzymes of carbohydoes not bind to the cytoskeleton, but instead binds drate metabolism, regulates the different pathways reversibly to mitochondria (see reviews 30–32). HK of glucose metabolism under different conditions. binds to porin at the contact sites between the mito-

Detachment of glycolytic enzymes from cytoskeleton of melanoma cells induced by calmodulin antagonists

Glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We have previously found that different calmodulin antagonists decrease the levels of allosteric activators of glycolysis, and reduce ATP content and cell viability in B16 melanoma cells. Here we report of a novel, additional, mechanism of action of calmodulin antagonists in melanoma cells. We show that these drugs cause a detachment of the glycolytic enzymes, phosphofructokinase (ATP: d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase ( d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of B16 melanoma cells. This effect was dose- and time-dependent, and preceded the decrease in cell viability. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton-membrane and would affect cytoskeleton structure. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by calmodulin antagonists, as well as their reported inhibitory action on cell proliferation, make these drugs most promising agents in treatment of cancer.

Glycolytic enzyme binding in Otala lactea hepatopancreas: effect of taxol, colchicine and cytochalasin B and D on the in vivo enzyme distribution.

The effect of cytoskeleton modulators on glycolytic enzyme binding was examined in the hepatopancreas of Otala lactea in an attempt to identify potential cellular binding sites. Binding was followed by measuring phosphofructokinase (PFK), aldolase (ALD), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) distribution between low speed pellets (12,000 xg), high speed pellets (100,000 xg) and high speed supernatants. Taxol (which stabilizes microtubules), colchicine (which destabilizes microtubules) and cytochalasin B and D (which destabilize F-actin filaments) were added to the homogenate prior to centrifugation. Addition of taxol increased the amount of PFK associated with the high speed pellet. Cytochalasin B and D reduced the binding of PFK and PK to the low speed pellet. ALD and GAPDH binding were unaffected by any treatment. Lowering the pH of the crude homogenate increased PFK binding to the low speed pellet by 33%. This effect could be reversed by addition of cytochalasin B and D suggesting that pH influences the PFK-F-actin interaction in vivo. The differential binding response of PFK, PK, ALD and GAPDH to added effectors suggests that, in the cell, PFK and PK are bound to different subcellular structural elements than are ALD and GAPDH.

A Glycolytic Enzyme Binding Domain on Tubulin

Cleavage of tubulin at tryptophan residues yielded several peptides, one of which strongly interacted with aldolase as determined by inhibition of aldolase activity. This peptide was identified as the C-terminal, residues 408–451, of the α-subunit of tubulin. Peptides with identical sequences to the C-terminal regions of the α- and β-subunits of tubulin were synthesized to further characterize interactions with glycolytic enzymes. A 43-amino-acid C-terminal peptide from α-tubulin (residues 409–451) was found to have binding properties similar to those of native tubulin and was designated the tubulin glycolytic enzyme binding domain (T-GEBD-43mer).

Glycolytic enzyme binding during entrance to daily torpor in deer mice (Peromyscus maniculatus).

Associations of glycolytic enzymes with the subcellular particulate fraction of skeletal muscle and heart were examined during entrance to daily torpor in deer mice (Peromyscus maniculatus). In skeletal muscle a significant decrease in enzyme binding occurred during torpor entrance for phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase, with an additional significant decrease for phosphofructokinase and pyruvate kinase during the deepest state of torpor. Reductions in enzyme binding during torpor entrance also occurred in heart; significant changes were observed in hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase binding. Contrary to the finding of additional reductions in enzyme binding seen in skeletal muscle, significant increases in enzyme binding during the deepest torpor state were observed for hexokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase in heart. These results suggest that a decrease in the binding of glycolytic enzymes to subcellular structures in skeletal muscle and heart may be at least partially responsible for initiating the reduction in metabolic rate during torpor entrance. This decreased binding may continue to mediate the metabolic reduction in skeletal muscle throughout torpor; heart, however, may require the use of different molecular mechanisms. The increased binding in heart during the deepest state of torpor may represent an anticipatory response in preparation for increased activity during arousal.

Free Radicals Induce Reversible Membrane-Cytoplasm Translocation of Glyceraldehyde-3-Phosphate Dehydrogenase in Human Erythrocytes

We investigated the role of oxygen free radicals in the modulation of glyceraldehyde-3-phosphate dehydrogenase binding to the erythrocyte membrane. Previous studies have demonstrated that in vitro tyrosine phosphorylation of Band 3 prevents the binding of various glycolytic enzymes to its cytoplasmic domain. Since these enzymes are inhibited in their bound state, the functional consequence of Band 3 tyrosine phosphorylation in red blood cells should be to increase glycolysis. To generate free radicals, we used an azocompound, the hydrophilic 2,2′-azobis(2-amidinopropane) hydrochloride, which, at 37°C and in the presence of oxygen, decomposes and produces peroxyl radicals at at constant rate. The reaction of peroxyl radicals with intact red cells induced a time-dependent loss of the membrane-bound glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, associated with a concomitant decrease in enzyme activity. At the same time, Band 3 was phosphorylated in tyrosine. These results were completely reversible in plasma after removal of the oxidative stress. The peroxyl radicals also enhanced the production of lactate in intact cells. Our data reveal a powerful mechanism of erythrocyte metabolic regulation that can boost or reduce energy production in times of special need such as during a free radical attack.