Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.     

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.     

Regulation of skeletal muscle metabolism by enzyme binding.

The random diffusion mechanism is usually assumed in analyzing the energetics of specific pathways despite the findings that enzymes associate with each other and (or) with various membranous and contractile elements of the cell. Successive glycolytic enzymes have been shown to associate in the cytosol as enzyme complexes or bind to the thin filaments. Furthermore, the degree of glycolytic enzyme interactions have been shown to change with altered rates of carbon flux through the pathway. In particular, the proportions of aldolase, phosphofructokinase, and glyceraldehyde phosphate dehydrogenase bound to the contractile proteins have been found to increase with increased rates of glycolysis. In addition, decreasing pH and ionic strength are also associated with an increase in glycolytic enzyme interactions. The kinetics displayed by interacting enzymes generally serve to enhance their catalytic efficiencies. The associations of the glycolytic enzymes serve to enhance metabolite transfer rates, increase the local concentrations of intermediates, and provide for regulation of activity via effectors. Therefore these interactions provide an additional mechanism for regulating glycolytic flux in skeletal muscle.     

In Saccharomyces cerevisiae,withdrawal of the carbon source results in detachment of glycolytic enzymes from the cytoskeleton and in actin reorganization

Metabolons are dynamic associations of enzymes catalyzing consecutive reactions within a given pathway. Association results in enzyme stabilization and increased metabolic efficiency. Metabolons may use cytoskeletal elements, membranes and membrane proteins as scaffolds. The effects of glucose withdrawal on a putative glycolytic metabolon/F-actin system were evaluated in three Saccharomyces cerevisiae strains: a WT and two different obligate fermentative (OxPhos-deficient) strains, which obtained most ATP from glycolysis. Carbon source withdrawal led to inhibition of fermentation, decrease in ATP concentration and dissociation of glycolytic enzymes from F-actin. Depending on the strain, inactivation/reactivation transitions of fermentation took place in seconds. In addition, when ATP was very low, green fluorescent protein-labeled F-actin reorganized from highly dynamic patches to large, non-motile actin bodies containing proteins and enzymes. Glucose addition restored fermentation and cytoskeleton dynamics, suggesting that in addition to ATP concentration, at least in one of the tested strains, metabolon assembly/disassembly is a factor in the control of the rate of fermentation.     

Localisation and quantification of dehydrogenase activities in single muscle fibres of mdx gastrocnemius

The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate subsarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions. Accepted: 12 October 1999     

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V _max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.     

Metabolic enzyme diversity in different human thyroid cell lines and their sensitivity to gravitational forces

Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase.     

Effect of anaerobiosis and anhydrobiosis on the extent of glycolytic enzyme binding in Artermia embryos

The effect of anaerobiosis and anhydrobiosis on the extent of binding of glycolytic enzymes to the particulate fraction of the cell was studied in Artemia salina embryos. During control aerobic development, trehalase, phosphofructokinase and pyruvate kinase showed an increase in the percentage associated with the particulate fraction which is consistent with the carbohydrate-based metabolism of Artemia embryos. However, anaerobiosis resulted in decreased enzyme binding for six glycolytic enzymes; hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase were the exceptions. Decreased enzyme binding was also observed after exposure to dehydrating conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms in Artemia embryos. This reversible interaction of glycolytic enzymes with structural proteins may account for part of the metabolic arrest observed during anaerobic dormancy and anhydrobiosis.Abbreviation pH_i intracellular concentration of H⁺ ions     

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.     

Evidence for glycolytic enzyme binding during anaerobiosis of the foot muscle ofPatella caerulea (L.)

Summary The effect of anaerobiosis and aerobic recovery on the degree of binding of glycolytic enzymes to the particulate fraction of the cell was studied in the foot muscle of the marine molluscP. caerulea, in order to assess the role of glycolytic enzyme binding in the metabolic transition between aerobic and anoxic states. Short periods of anoxia (2 h, 4 h) resulted in an increase in enzyme binding in association with the increased glycolytic rate observed; this was particularly pronounced for phosphorylase, phosphofructokinase, aldolase, pyruvate kinase and lactate dehydrogenase. Decreased enzyme binding was observed after prolonged periods of anoxia. These effects were reversed and control values re-established when animals were returned to aerobic conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms inP. caerulea foot muscle. This reversible interaction of glycolytic enzymes with structural proteins may constitute an additional mechanism for metabolic control.     

Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach

In order to identify target antigens of anti-endothelial cell (anti-EC) antibodies (AECA) in healthy individuals, we have used a proteomic approach combining 2-DE and immunoblotting. Whole cell protein extracts obtained from human umbilical vein EC (HUVEC) cultures were used as a source of antigens. Serum IgG from 12 healthy blood donors were tested at a concentration of 200 microg/mL. Targeted spots were identified by MS. The HUVEC proteome was composed of 884 protein spots. Among these, 61 +/- 25.8 (mean +/- SD) spots were recognized by serum IgG from healthy individuals, with marked differences from one individual to another. Among these spots, 11 were recognized by serum IgG from all healthy individuals tested. These spots corresponded to six different proteins with several spots corresponding to different isoforms of the same protein. Target antigens were: cytoskeletal proteins (beta-actin, alpha-tubulin, and vimentin); glycolytic enzymes (glucose-3-phosphate-deshydrogenase and alpha-enolase); and prolyl-4-hydroxylase beta subunit, a member of the disulfide isomerase family. This study shows that the repertoire of IgG AECA is heterogeneous among healthy individuals. IgG from all of the healthy individuals tested recognized a restricted set of highly conserved ubiquitous proteins playing key roles in cell biology and maintenance of homeostasis.     

Glyceraldehyde-3-phosphate dehydrogenase associates with actin filaments in serum deprived NIH 3T3 cells only

The in vitro interaction between the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytoskeletal elements is well documented. To verify this association within cells, the intracellular distribution of GAPDH under various metabolic conditions has been investigated in immunostained cells or cells expressing GAPDH as a GFP fusion protein. GAPDH was homogeneously distributed in the cytoplasm and no interaction of GAPDH with cytoskeletal elements, neither with microfilaments nor microtubules or intermediate filaments, was detectable. In living cells expressing GFP-GAPDH, stress fibres were excluded from the fluorescence. In contrast to proliferating cells, the cytoplasmic GAPDH of serum-depleted cells was not homogeneously distributed, but colocalised with stress fibres. The mechanism for stimulating this actin-binding affinity was independent of the NO-signalling pathway. The results support the idea of a specialised function for the interaction of GAPDH and cytoskeletal elements, rather than a general function, as e.g. microcompartmentalization of glycolytic enzymes.     

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.     

Supramolecular organization of glycolytic enzymes

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscle and to erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites the structure of glycolytic enzyme complex adsorbed to a biological support has been proposed. The key role in the formation of the multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex fixed on structural proteins of skeletal muscle contains one tetrameric molecule of 6-phosphofructokinase and at two molecules of other glycolytic enzymes. Hexokinase is not involved in the complex composition. The molecular mass of the multienzyme complex is about 2,6 X 10(6) Da. The formation of the multienzyme complex leads to the compartmentation of the glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.     

Experimental determination of control of glycolysis in Lactococcus lactis

The understanding of control of metabolic processes requires quantitative studies of the importance of the different enzymatic steps for the magnitude of metabolic fluxes and metabolite concentrations. An important element in such studies is the modulation of enzyme activities in small steps above and below the wild-type level. We review a genetic approach that is well suited for both Metabolic Optimization and Metabolic Control Analysis and studies on the importance of a number of glycolytic enzymes for metabolic fluxes in Lactococcus lactis. The glycolytic enzymes phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis, but it cannot yet be excluded that one of the remaining glycolytic steps is a rate-limiting step for the glycolytic flux.     

Pressure-adaptive differences in lactate dehydrogenases of three hagfishes: <Emphasis Type="Italic">Eptatretus burgeri,Paramyxine atami</Emphasis> and <Emphasis Type="Italic">Eptatretus okinoseanus</Emphasis>

The tolerance of abyssal pressures likely depends on adaptive modifications of fish proteins. However, structural modifications of proteins which allow functioning at high pressure remain unclear. We compared the activities of lactate dehydrogenase (LDH), an important enzyme in glycolytic reaction, in three hagfishes inhabiting different depths under increased pressure. LDH in Eptatretus okinoseanus, found at a depth of 1,000 m, was highly active at high pressure of 100 MPa maintaining the activity at 70% of that at 0.1 MPa. In contrast, LDH activity in Paramyxine atami, found at 250–400 m, decreased to 55% at 15 MPa, and that in Eptatretus burgeri, found at 45–60 m, was completely absent at 5 MPa. The result suggests that subunit interaction of the LDH-tetramer is more stable in E. okinoseanus than that in P. atami and E. burgeri under high-pressure conditions. We found six amino acid substitutions between the three LDH primary structures. Accordingly, these amino acid residues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions.     

Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.     

Interaction of lactate dehydrogenase with structural cell components: possible physiological significance]

Reversible binding of cytoplasmic enzymes to structural elements of the cell is one of mechanisms of in vitro regulation of enzyme properties. The results on lactate dehydrogenase interactions with myofibrillar proteins and membranes and the changes in enzyme properties induced by these interactions (modification of kinetic parameters and stability) are analyzed. A hypothesis is proposed concerning the functional role of reversible lactate dehydrogenase interactions with structural components of the cell during glycolysis activation.