Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.     

Reassessment of the cold‐labile nature of phosphofructokinase from a hibernating ground squirrel

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37°C). A 78 ± 2.5% loss of PFK activity during 1 h incubation at 5°C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 ± 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5°C also indicated that ~ 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.     

Glycolytic enzyme interactions with yeast and skeletal muscle F-actin

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

The effect of fatigue on the binding of glycolytic enzymes in the isolated gastrocnemius of Rana pipiens

Fatigue of isolated gastrocnemius muscles from R. pipiens leads to a marked increase in the proportion of phosphofructokinase bound to the particulate fraction and a decrease in the binding of lactate dehydrogenase, pyruvate kinase, creatine phosphokinase and glyceraldehyde-3-phosphate dehydrogenase. Only the proportion of aldolase bound to the particulate fraction was unaffected by fatigue. This pattern was unchanged when fatigued muscles were extracted at pH 6.5 rather than 7.5. Thus, muscle fatigue leads to opposite changes in the binding of the glycolytic enzymes.     

Studies on the topographical localization of the binding sites for substrate and for actin on the enzymes, glyceraldehydephosphate dehydrogenase and phosphofructokinase

The effect of proteolysis on the catalytic activity and the binding capacity for actin has been studied in the case of both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase (PFK). With both of these enzymes, the differential response of these two parameters is interpreted as an indication of the distinct topographical separation of the active sites and binding sites. These results have been discussed in relation to the positioning of the catalytic and binding sites on these enzymes, the nature of their interaction with actin, their relative stability in cellular situations and the phenomenon of enzyme ambiguity.     

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.     

Activity patterns of phosphofructokinase,glyceraldehydephosphate dehydrogenase,lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle

Summary Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Phosphofructokinase activity and acidosis during short-term tetanic contractions.

Anaerobic energy production is essential for the production of muscular tension when the demand for energy is greater than can be provided aerobically and when oxygen is in short supply. The largest source of anaerobic energy is from the glycolytic pathway. With sustained tetanic contractions, muscle glycolytic activity is high and hydrogen ions (H+) accumulate while tension production decreases. The increasing [H+] and decreasing tension led to the suggestion that H+ inhibits the activity of the regulatory glycolytic enzyme phosphofructokinase (PFK). Early in vitro work confirmed the H+ sensitivity of PFK in the test tube, indicating that little PFK activity should persist at a pH of 6.9-7.0. However, in situ and in vivo experiments suggested that significant PFK activity was maintained during intense contractions when muscle pH decreased to 6.4-6.6. There are several concerns associated with the application of in vitro findings to in vivo exercise situations: (i) there is little in vitro work in mammalian skeletal muscle with substrate and modulator concentrations representative of exercise, (ii) most in vitro analyses of PFK activity are performed following the dilution of the enzyme in mediums with low protein concentration, and (iii) do the modulators identified in vitro exist in high enough in vivo concentrations at rest and during exercise to contribute to the regulation of PFK? More recent in vitro and in situ PFK experiments have overcome some of these concerns. They confirm that during intense, short-term tetanic contractions, PFK activity is well matched to the ATP demand despite decreases in pH to approximately 6.4-6.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reversible binding of glycolytic enzymes in ovine skeletal muscle in response to tetanic stimulation

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in sheep hind muscles after electrical stimulation. As compared to the control muscles, stimulation led to significant increases in the amount of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase bound to the particulate fraction. The bindng of other glycolytic enzymes was not significantly altered. A servey of different hind limb muscles at variable rates of stimulation revealed that each muscle exhibited its own characteristic response pattern in terms of the level of increased enzyme binding. Generally, an increased stimulation rate led to greater enzyme adsorption. The increase in enzyme binding was rapidly reversible for it was shown that the amount of enzyme bound quickly returned to control values when the muscles were allowed to recover in the live anaesthetised animal following cessation of stimulation. Those muscles which exhibited increased enzyme binding were characterised by a marked loss of glycogen and accumulation of lactate suggesting that accelerated glycolytic flux was a necessary condition for the observation of increased enzyme binding. In support of this, enzyme adsorption was observed to be greatest on stimulation of ischemic muscles, whereas in trained muscles, or muscles with depleted glycogen stores induced by prior adrenalin treatment, the increased enzyme binding response was greatly diminished. It is concluded that the variable binding of key glycolytic enzymes has a role to play in the regulation of glycolytic behaviour in skeletal muscle.     

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.     

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.     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

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

Evidence for the short-term regulation of glycolytic flux in the isolated perfused ventricle of the land snail Helix lucorum (L.) after treatment with serotonin (5-hydroxytryptamine)

The glycolytic flux and the regulation of phosphofructokinase (PFK) activity by fructose 2,6-bisphosphate and covalent modification was investigated in isolated ventricles of land snail Helix lucorum perfused with or without serotonin. Serotonin evoked a significant increase in the level of glycolytic intermediates and a threefold increase of glycolytic flux. Studies of saturation curves of PFK for the substrate fructose 6-phosphate at pH similar to intracellular pH of heart muscle showed that serotonin increases enzyme sensitivity to activation by fructose 6-phosphate. Moreover, PFK preparations from ventricles perfused with serotonin exhibited lower K _a values for the activators AMP and fructose 2,6-bisphosphate, compared with the enzyme preparations from serotonin-untreated ventricles. The results suggest that PFK was converted to a more active form when exposed to serotonin. In vitro experiments of PFK phosphorylation showed that the conversion of the enzyme to a more active form was possibly due to its phosphorylation by an endogenous cyclic-AMP-dependent protein kinase. The concentration of fructose 2,6-bisphosphate increased in serotonin-treated ventricles and it exerted a synergistic effect with AMP on the activation of PFK. The bound fraction of glycolytic enzymes increased in the serotonin-treated ventricles only after the 4th min of perfusion. The results suggest that the stimulation of glycolytic flux in the ventricles of H. lucorum in the first minutes of perfusion with serotonin was partly due to the activation of PFK via enzyme molecule covalent modification and to increase of fructose 2,6-bisphosphate. Accepted: 8 April 1997     

Intracellular glucose and binding of hexokinase and phosphofructokinase to particulate fractions increase under hypoxia in heart of the amazonian armored catfish (Liposarcus pardalis)

Armored catfish (Liposarcus pardalis), indigenous to the Amazon basin, have hearts that are extremely tolerant of oxygen limitation. Here we test the hypothesis that resistance to hypoxia is associated with increases in binding of selected glycolytic enzymes to subcellular fractions. Preparations of isolated ventricular sheets were subjected to 2 h of either oxygenated or hypoxic (via nitrogen gassing) treatment during which time the muscle was stimulated to contract. The bathing medium contained 5 mM glucose and was maintained at 25 degrees C. Initial experiments revealed increases in anaerobic metabolism. There was no measurable decrease in glycogen level; however, hypoxic treatment led to a twofold increase in heart glucose and a 10-fold increase in lactate content. It is suggested that the increase in heart glucose content is a result of an enhanced rate of facilitated glucose transport that exceeds the rate of phosphorylation of glucose. Further experiments assessed activities of metabolic enzymes in crude homogenates and subsequently tracked the degree of enzyme binding associated with subcellular fractions. Total maximal activities of glycolytic enzymes (hexokinase [HK], phosphofructokinase [PFK], aldolase, pyruvate kinase, lactate dehydrogenase), and a mitochondrial marker, citrate synthase, were not altered with the hypoxic treatment. A substantial portion (>/=50%) of HK is permanently bound to mitochondria, and this level increases under hypoxia. The amount of HK that is bound to the mitochondrial fraction is at least fourfold higher in hearts of L. pardalis than in rat hearts. Hypoxia also resulted in increased binding of PFK to a particulate fraction, and the degree of binding is higher in hypoxia-tolerant fish than in hypoxia-sensitive mammalian hearts. Such binding may be associated with increased glycolytic flux rates through modulation of enzyme-specific kinetics. The binding of HK and PFK occurs before any significant decrease in glycogen level.     

Skeletal muscle glycogenolysis, glycolysis, and pH during electrical stimulation in men

Glycogenolytic and glycolytic rates were estimated and muscle pH (pHm) was measured in electrically stimulated quadriceps femoris muscles of seven men. Leg blood flow was occluded and muscles were stimulated 64 times at 20 Hz, with contractions lasting 1.6 s and separated by pauses of 1.6 s. Muscle biopsies were obtained at rest and following 16, 32, 48, and 64 contractions. Glycolytic intermediates and several modulators of the glycolytic enzyme phosphofructokinase (PFK) were measured. Glycogenolytic and glycolytic rates were 1.68 and 1.26 mmol glucosyl units X kg dry muscle-1 X S-1 contraction time during the initial 16 contractions and pHm decreased from 7.00 +/- 0.01 to 6.70 +/- 0.03. During the subsequent 32 contractions both glycogenolytic and glycolytic rates were maintained at approximately 0.70 mmol X kg-1 X S-1 and pHm decreased to 6.45 +/- 0.04. In the final 16 contractions, both rates were very low and pHm was unchanged. Therefore, PFK remained active despite increasing acidity until pHm decreased to approximately 6.45. We conclude that increases in the concentrations of several positive modulators partially reverses pH-dependent ATP inhibition of PFK in vivo, permitting glycolytic activity to continue in the pHm range of 6.70-6.45.     

Decrease in cytoskeleton-bound phosphofructokinase in muscle induced by high intracellular calcium, serotonin and phospholipase A2 in vivo

1. Particulate (cytoskeleton-bound) and soluble phosphofructokinase (PFK), separated from rat muscle, exhibited different allosteric properties; in contrast to the soluble PFK, the bound enzyme was not sensitive to allosteric regulation. 2. Treatment of muscle with Ca2(+)-ionophore A23187, serotonin, or phospholipase A2, reduced the binding of PFK and aldolase. 3. The decrease in enzymes' binding was most probably mediated by the rise in free intracellular Ca2+ induced by these agents, as we found that direct addition of Ca2+ to the particulate fraction of muscle, caused solubilization of bound PFK and aldolase. 4. The reduction in binding of PFK and aldolase to cytoskeletal proteins, may have a deleterious effect on muscle function and structure, and may be involved in the mechanism of muscle damage in pathological conditions where accumulation of Ca2+ occurs.     

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.     

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.     

Effects of m-Cl-peroxy benzoic acid on glycolysis in Saccharomyces cerevisiae

Concentrations of m-Cl-peroxy benzoic acid (CPBA) higher than 0.1 mM decrease the ATP-content of Saccharomyces cerevisiae in the presence of glucose in 1 min to less than 10% of the initial value. In the absence of glucose, 1.0 mM CPBA is necessary for a similar effect. After the rapid loss of ATP in the first min in the presence of glucose caused by 0.2 mM CPBA, the ATP-content recovers to nearly the initial value after 10 min. Aerobic glucose consumption and ethanol formation from glucose are both completely inhibited by 1.0 mM CPBA. Assays of the activities of nine different enzymes of the glycolytic pathway as well as analysis of steady state concentrations of metabolites suggest that glyceraldehyde-3-phosphate dehydrogenase is the most sensitive enzyme of glucose fermentation. Phosphofructokinase and alcohol dehydrogenase are slightly less sensitive. Incubation for 1 or 10 min with concentrations of 0.05 to 0.5 mM CPBA causes a) inhibition of glyceraldehyde-3-phosphate dehydrogenase, b) decrease of the ATP-content and c) a decrease of the colony forming capacity. From these findings it is concluded that the disturbance of the ATP-producing glycolytic metabolism by inactivation of glyceraldehyde-3-phosphate dehydrogenase may be an explanation for cell death caused by CPBA.Abbreviations CPBA m-Chloro-peroxy benzoic acid - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - F-1,6-P₂ frnctose-1,6-bisphosphate - DAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - 2PGA 2-phosphoglycerate - PEP phosphoenol pyruvate - Pyr pyruvate - EtOH ethanol - PFK phosphofructokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - ADH alcohol dehydrogenase Dedicated to Prof. Dr. Wolfgang Gerok at the occasion of his 60th birthday