The influence of deoxyribonuclease I and cytochalasin D on the release of glycolytic enzymes from digitonized cells

In permeabilized cells, deoxyribonuclease I has been demonstrated to cause a decrease in the extent of binding to cellular structure of all of the glycolytic enzymes other than phosphofructokinase, with this decrease being most marked for aldolase and glyceraldehydephosphate dehydrogenase. Cytochalasin D, in contrast, did not produce this type of effect. These results have been discussed in relation to the evidence for the existence of a complex of glycolytic enzymes which binds to elements of the cytoplasmic matrix, and the possible organization of this complex.     

The influence of molecular crowding on the binding of glycolytic enzymes to cytoskeletal structure

In a study of the interactions between glycolytic enzymes and cytoskeletal structure, the effect of increasing the degree of molecular crowding by the addition of protein was studied. This treatment resulted in an increased degree of binding of all the glycolytic enzymes, but with the most marked increases occurring with phosphofructokinase, enolase and pyruvate kinase. The significance of this data has been discussed in relation to the relative affinities of interaction of the individual components, the influence of molecular crowding and the physiological significance of this phenomenon.     

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.     

The influence of fructose-1:6-bisphosphate on the release of glycolytic enzymes from cellular structure

In order to provide information on the relative binding characteristics of glycolytic enzymes, the effect of fructose-1,6-bisphosphate (FBP) on the release of glycolytic enzymes from cultured pig kidney cells treated with digitonin has been studied. In the absence of FBP, a differential release of these enzymes was observed, with the order of retention being aldolase greater than glyceraldehyde-3-phosphate dehydrogenase greater than glucosephosphate isomerase, triosephosphate isomerase, phosphoglycerokinase, phosphoglucomutase, lactate dehydrogenase, enolase, pyruvate kinase and phosphofructokinase. In the presence of fructose-1,6-bisphosphate, the release of aldolase was considerably enhanced, whereas the release of phosphofructokinase and pyruvate kinase was decreased by this metabolite. No significant alterations in the rate of release of the other enzymes was caused by FBP. These data have been discussed in relation to their contribution to the knowledge of the degree of association and order of binding between glycolytic enzymes and the cytoplasmic matrix.     

Microenvironmental factors and the binding of glycolytic enzymes to contractile filaments.

1. In reviewing the microenvironmental factors involved in the binding of the glycolytic enzymes to contractile filaments, consideration has been given to the significance of molecular crowding in maintaining these interactions under cellular conditions, and the influence of hormones, metabolites, pH and enzyme modifications on these phenomena. 2. Overall, these data serve to emphasize the biological reality of these associations, and their micro-organizational adaptations during physiological activities.     

Chemical modification of the actin binding site of rabbit muscle aldolase by diethylpyrocarbonate

Summary To extend the available information on the significance of the interactions between glycolytic enzymes and the actin component of the cellular ultrastructure, investigations into the compositional characteristics of the actin binding site on one of the major glycolytic enzymes, aldolase, have been undertaken. As the electrostatic nature of the association has been previously reported indicative of a cationic region on the enzyme involved in the binding, these studies have investigated the possibility of the involvement of histidine residues in this binding region. By the use of the histidine specific reagent, diethylpyrocarbonate, we have been able to establish a difference in nature of an actin binding domain and the active site domain which does contain an essential histidine. The results have been discussed in relation to the significance of this finding with respect to the binding of aldolase to subcellular structure.     

Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle

Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pKa of 7.3 in 30 mM imidazole. PFK binding increased at lower pH values; at 150 mM KCl the apparent pKa value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium--with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F.D. Shaw, and D.J. Morton (1980) Biochem. J. 186, 105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.     

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.     

Association of rabbit muscle glycolytic enzymes with filamentous actin. A counter-current distribution study at high ionic strength

The association between purified glycolytic enzymes and filamentous actin from rabbit muscle has been studied by counter-current distribution. The co-distribution of a glycolytic enzyme and filamentous actin leads to a significant change in the counter-current distribution profile of the enzyme whereas that of actin is unaffected. The changes in the distribution profiles clearly demonstrated that all glycolytic enzymes studied, though to different extents, bind to filamentous actin. The aqueous two-phase system used for the studies contained dextran, poly(ethyleneglycol) and 150 millimolal potassium phosphate buffer, pH 7.0. Since the ionic strength of the two-phase system is determined mainly by the buffer, the glycolytic enzymes are evidently able to associate with filamentous actin, at least in the presence of neutral polymers, at ionic strengths comparable to or higher than those assumed to prevail in vivo.     

The association of glycolytic enzymes with cellular and model membranes

This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity     

On the existence of a complex of glycolytic enzymes

It has been suggested in the literature that the glycolytic enzymes are organized into a multi-enzymic complex. We have evaluated this hypothesis for the phosphotriose-glycerate phosphate group of glycolytic enzymes of muscle using sucrose density gradient centrifugation, gel filtration, and ultrafiltration. Attempts were made to avoid dilution and changes in pH. The ratio of activities of the phosphotriose-glycerate phosphate group of enzymes was similar to that found in several other tissues that has led to their designation as a constant proportion group of enzymes. However, no evidence was obtained that they exist as a multi-enzymic complex in chicken breast muscle. As the pH of the press juice is raised to 7.0 and the temperature to 25°C, association occurs between some components in the muscle press juice as evidenced by a blocking of the pores of an ultrafiltration membrane. This association, however, does not involve the enzymes of the phosphotriose-glycerate phosphate group.     

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.     

Interactions between glycolytic enzymes and cytoskeletal structure--the influence of ionic strength and molecular crowding

In a study of the interactions between glycolytic enzymes and cytoskeletal structure, the effect of increasing the degree of molecular crowding by the addition of physiological concentrations of saline and protein was studied. Increasing the ionic strength to physiological levels resulted in only a slight decrease in the retention of most of enzymes, whereas the establishment of physiological concentrations of both saline and protein, caused a markedly increased degree of binding of all the glycolytic enzymes. The implications of this data have been discussed in relation to the relative affinities of interaction of the individual components, the influence of molecular crowding and the physiological significance of this phenomenon.     

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.     

Equilibrium partition studies of the myofibrillar interactions of glycolytic enzymes

The interactions of several glycolytic enzymes with muscle myofibrils in imidazole-chloride buffer (pH 6.8, I 0.158) have been investigated by equilibrium partition studies. Results for aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and phosphofructokinase are interpreted in terms of a myofibrillar capacity of 76 nmol/g protein and a single intrinsic association constant for each tetravalent enzyme with matrix sites. The existence of separate myofibrillar sites for aldolase and glyceraldehyde-3-phosphate dehydrogenase is established by demonstrating independence of the binding of each enzyme upon the presence of the other. Although this investigation provides further physicochemical support for myofibrillar adsorption of glycolytic enzymes in the cellular environment, its findings are incompatible with the proposition (B. I. Kurganov, N. P. Sugrobova, and L. S. Mil'man (1985) J. Theor. Biol. 116, 509-526) that the phenomenon reflects the formation of a specific multienzyme complex attached to the myofibril.     

Adsorption of peripheral enzymes to membrane anchor proteins

The character of the isotherms of specific adsorption of peripheral enzymes to dimeric anchor proteins embedded in the membrane has been analysed. The situations are discussed when adsorption corresponds to the stoichiometry of one or two molecules of peripheral enzyme per dimeric binding site. The corresponding expressions describing the competitive interrelationships between peripheral enzymes adsorbed to the same binding sites have been derived. The experimental data on the adsorption of glycolytic enzymes to erythrocyte membranes are used for the illustration of the theoretical predictions. The physiological role of enzyme self-association which leads to the formation of enzyme oligomers of unlimited length is discussed. It is assumed that under in vivo conditions the association sites of such enzymes are saturated through interactions with anchor proteins of subcellular structures and with the enzymes of the corresponding metabolic pathways. Therefore the linearly associating enzymes play the key role in the formation of multienzyme complexes attached to subcellular structures. The significance of 6-phosphofructokinase adsorption to erythrocyte membranes in the formation of the complex of glycolytic enzymes is discussed.     

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.     

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.     

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.