Association Of Glycolytic Enzymes Research Articles

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

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.

The association of glycolytic enzymes from yeast confers resistance against inhibition by trehalose

During stress, many organisms accumulate compatible solutes. These solutes must be eliminated upon return to optimal conditions as they inhibit cell metabolism and growth. In contrast, enzyme interactions optimize metabolism through mechanisms such as channeling of substrates. It was decided to test the (compatible solute) trehalose-mediated inhibition of some yeast glycolytic pathway enzymes known to associate and whether inhibition is prevented when enzymes are allowed to associate. Trehalose inhibited the isolated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hexokinase (HXK), but not aldolase (ALD) nor phosphoglycerate kinase (PGK). When these enzymes were mixed in pairs, both GAPDH and HXK were protected by either ALD or PGK acquiring the inhibition behavior of the resistant enzyme. GAPDH was not protected by HXK, albumin or lactate dehydrogenase (LDH). Also, ALD did not protect glucose 6-phosphate dehydrogenase (G6PDH), suggesting that protection is specific. In yeast cell extracts, fermentation was resistant to trehalose inhibition, suggesting all enzymes involved in the glucose-dependent production of ethanol were stabilized. It is suggested that during the yeast stress response, enzyme association protects some metabolic pathways against trehalose-mediated inhibition.

Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells.

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

Dependence of energy metabolism on the density of cells in culture.

Metabolism in cultured cells strongly depends on cell density, thus cell density may be an important factor in biotechnological maintenance of cell cultures. Therefore the energy metabolism of three related cell types with different proliferation activity has been characterised: density controlled primary or secondary fibroblasts [pmf], immortalized, but still density controlled 3T3 cells, and SV40-transformed 3T3 lacking growth control. The investigations revealed the decrease of oxygen consumption, net lactate production, ATP-content, NAD-content and NAD-redox potential, and F-actin content with increasing culture density in pmfs, less distinct in 3T3 cells and in SV40-3T3 cells. The main decrease of these factors is related to cell-cell contacts rather than to proliferation which ceases at least two division cycles after most cells contacted a neighbouring cell. SV40-3T3 cells also at preconfluent densities exhibit relatively low metabolic activity as revealed by the above mentioned factors. Supply with metabolites for catalytic processes seems to be the rate limiting factor as deduced from a decreasing NADH/total NAD ratio. SV40-3T3 cells lack a contact mediated reduction of energy metabolism which is in accord with the missing contact inhibition of motility and proliferation in SV40-3T3 cells. Because of a possible association of glycolytic enzymes with actin, F-actin content has been determined. No correlation, however, was revealed by the F-actin-/lactate ratio. This may be due to the fact that about 50% of the lactate released into the culture medium originated to from glutaminolysis rather than glycolysis. Only in SV40-3T3 cells were respiration and lactate production insensitive to glutamine deprivation and in these cells both these parameters did not change significantly, therefore they did not allow testing as to whether f-actin content and glycolytic activity are correlated. Fractionated release of adeninnucleotides revealed that energy charge in the cytosolic fraction is the only factor which strictly correlates with the cessation of proliferation. Thus energy charge of the cytosol seems to be the only factor of these studied here, which can be used as an indicator of a culture's commitment for further proliferation. To compare energy metabolic parameters among different cell types one always has to take into account the density dependence of these factors.

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.

Insulin activation of NADH ferricyanide reductase in human erythrocytes is mediated by the insulin receptor tyrosine kinase: a comparative study in normal and diabetic states.

We have previously reported that NADH ferricyanide reductase in human erythrocytes is stimulated by insulin. Hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors like vanadate, indicating the involvement of glycolysis in the mechanism by which insulin stimulates ferricyanide reduction. Activation of erythrocyte metabolism in response to insulin could be a result of hormone binding to its receptor, inducing phosphorylation of band 3 (at a site for reversible association of glycolytic enzymes) and/or other membrane proteins like the Na(+)/H(+) antiport. Activation of the antiporter protein by insulin can stimulate glycolysis by an increase in intracellular pH, an effect which is prevented by amiloride. Evidence for a role for tyrosine phosphorylation in triggering the reductase activation came from studies with protein kinase inhibitors. Genistein, sphingosine and acridine orange have been shown to prevent insulin-stimulated ferricyanide reduction, implicating tyrosine phosphorylation as an important signal for activation of the enzyme by insulin. To evaluate activation of the enzyme by insulin stimulated phosphorylation, a comparative study was done using erythrocytes from healthy and diabetic humans. We measured ferricyanide reductase activities in basal and insulin stimulated states. Basal activities were lower in diabetics than in normal humans. Nevertheless, hormone stimulated activities were similar, despite earlier reports of decreased receptor phosphorylation of exogenous substrates in type 2 diabetics. These observations, together with previous ones, suggest that insulin-receptor kinase interaction may mediate the action of insulin on human erythrocytes by phosphorylation of cellular proteins like band 3 and/or the Na(+)/H(+) antiport.

Glycolytic and Associated Enzymes of Rainbow Trout (Oncorhynchus Mykiss) Red Cells: In Vitro And In Vivo Studies

ABSTRACT Studies were undertaken in vitro and in vivo to assess the maximal activities of 26 glycolytic and associated enzymes of rainbow trout (Oncorhynchus mykiss) red cells. The red cells possess a complete sequence of glycolytic enzymes capable of anaerobic oxidation of glucose to lactate. Red cell pyruvate kinase (PK) was inhibited by ATP (I50 ≈ 5 mmol I 1−1), but was not sensitive to alanine inhibition or fructose-1,6-bisphosphate activation. The properties of red cell PK were similar to those of the muscle-type enzyme. Lactate dehydrogenase (LDH) from the trout erythrocyte resembled LDH from trout heart in terms of pyruvate inhibition in vitro. Enzymes associated with phosphagen and amino acid metabolism as well as the pentose phosphate shunt were also present. However, enzyme indicators of glycogenolytic and gluconeogenic potential were either absent or present at very low levels. The capacity for aerobic respiration via the tricarboxylic acid (TCA) cycle was suggested by the presence of citrate synthase activity. The association of glycolytic enzymes with the particulate fraction of the red cell was assessed for six enzymes. No binding was detected for hexokinase, PK and LDH; low levels of binding by phosphofructokinase (5%), aldolase (1%) and glyceraldehyde-3-phosphate dehydrogenase (3%) were not altered by strenuous exercise. Glycolytic enzyme binding, therefore, does not appear to be an important regulator of energy metabolism in these cells. Intracellular glucose levels, in contrast, appeared to be regulated following exercise stress. An increase in intra-erythrocytic lactate levels at this time may reflect the importance of exogenously produced lactate as a substrate for ATP production. The description of trout red cell metabolism presented here provides a basis for further study of the relationships between organismic gas exchange and molecular-level adaptation of nucleated red cell function.

Spreading of trypsinized cells: Cytoskeletal dynamics and energy requirements

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothelia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae. The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2-4-(dimethylamino)-styryl-1-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3] under conditions of high ATP content. In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3]; motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network. During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2(+)-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.

Association of glycolytic enzymes with the erythrocyte membrane.

Association of glycolytic enzymes with the erythrocyte membrane.

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.

Association of glycolytic enzymes with particulate fractions from nerve endings

Several glycolytic enzymes were observed to have between 40–90% of their activities associated with the particulate fractions of lysed nerve endings. The enzymes showing high particulate activity in lysed nerve endings were hexokinase (EC 2.7.1.1), aldolase (EC 4.1.2.13), glucosephosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase (EC1.2.27), prruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.27). With the exception of phosphofructokinase, 80% or more of the particle associated activity of each enzyme was solubilized by salt treatment indicating the association with particles was ionic. Sub-fractionation of lysed nerve endings showed hexokinase and fumarase (EC 4.2.1.2) had the highest specific activity in the same fractions which is consistent with observations indicating that hexokinase is associated with mitochondria. The other glycolytic zymes having high particulate activity, aldolase, glucosephosphate isomerase, phosphofructokinase, glyceraldehyde-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, showed enrichment in fractions containing synaptosomal membranes, i.e. the fractions having highest specific activity of acetylcholinesterase (EC 3.1.1.7) and (Na + + K +-ATPase (EC 3.6.1.3).

On the association of glycolytic enzymes with structural proteins of skeletal muscle

1. 1. The effects of protein concentration and ionic strength on the adsorption of the individual glycolytic enzymes to F-actin and F-actin—trypomyosin—troponin have been studied. 2. 2. Appreciable association was demonstrated under conditions of physiological ionic strength and high protein concentration, and tropomyosin—troponin established as an important and generalized component of these interactions. 3. 3. Phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate isomerase were strongly bound under these conditions, while triosephosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and hexokinase displayed less adsorption to the structural proteins. 4. 4. The influence of a number of parameters on the adsorption phenomena was examined. Ca 2+ and fructose 1,6-diphosphate increased the adsorption of aldolase, lactate dehydrogenase and pyruvate kinase, while decreasing the adsorption of the enzymes of the constant-proportion group. 5. 5. Of the other major enzymic components of skeletal muscle, creatine kinase, adenylate kinase and malate dehydrogenase showed no adsorption to F-actin—tropomyosin—troponin under the experimental conditions. Some adsorption was evident, however, in the case of aspartate aminotransferase, (NADP) isocitrate dehydrogenase and α-glycerolphosphate dehydrogenase. 6. 6. These results have been discussed in relation to their functional significance and the roles of enzyme compartmentation in the cell.

On the association of glycolytic components in skeletal muscle extracts

1. 1. In an extension of previous studies which demonstrated the existence of an association of glycolytic components in extracts of rat skeletal muscle, the properties of this complex have been further investigated. 2. 2. It has been shown that this association of glycolytic enzymes may also be observed in extracts of ovine muscle under physiological conditions of pH and ionic strength, and that the association is sensitive to variations of pH and the concentrations of proteins and specific metabolites such as ATP and fructose 1,6-diphosphate. Furthermore, myosin was identified as a necessary component for complex formation under the experimental conditions. 3. 3. The size of the observed complex of glycolytic enzymes increases with increasing protein concentration; at 52 mg protein/ml, s 20,w values of over 100 are indicated. 4. 4. These data have been discussed in relation to the possible physiological significance of the multi-enzyme association.