Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.     

In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis.

Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates.Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene.

A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3-phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde-3-phosphate dehydrogenases. According to the irreversible catabolic character of the non-phosphorylating reaction, the GapN-complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden-Meyerhof pathway with no net energy yield is feasible.     

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.     

Fluorescence studies on glyceraldehyde-3-phosphate dehydrogenase from bovine heart muscle

Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that catalyses conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. ATP has been found to have an inhibitory effect on this enzyme. To establish the interaction between the enzyme and ATP, a fluorescence technique was used. Fluorescence quenching in the presence of ATP suggests cooperative binding of ATP to the enzyme (the Hill obtained coefficient equals 2.78). The interaction between glyceraldehyde-3-phosphate dehydrogenase and ATP may control not only glycolysis but other activities of this enzyme, such as binding to the cytoskeleton.     

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     

Synthesis of adenosine triphosphate by myelin of spinal nerves of rabbit

Abstract—

• 1 The myelin fraction isolated by isopycnic gradient centrifugation from rabbit nerve is able to synthesize ATP at substrate level through the Embden-Meyerhof pathway. Suitable conditions are described to preserve the association of glycolytic enzymes with isolated myelin.
• 2 Except for phosphofructokinase and ketose-1-phosphate aldolase, all the remaining glycolytic enzymes are present in the myelin. A wide divergence was found in the firmness of the association of individual glycolytic enzymes with myelin under the condition of isolation; some, like glucosephosphate isomerase and glyceraldehydephosphate dehydrogenase were retained in high percentage (about 60 per cent of the activity of the homogenate is myelin-bound); others were weakly bound (no more than 7–6 percent of the lactate dehydrogenase activity of the homogenate is myelin-bound).
• 3 By using glyceraldehyde-3-phosphate as substrate for glycolysis, about 25 per cent of the total glycolytic activity of rabbit-nerve homogenate is associated with the myelin.
• 4 Glucosephosphate isomerase and lactate dehydrogenase may be extracted from and readily recombined with the myelin.

     

Glycation,glycolysis, and neurodegenerative diseases: Is there any connection?

This review considers the interrelation between different types of protein glycation, glycolysis, and the development of amyloid neurodegenerative diseases. The primary focus is on the role of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase in changing the concentration of carbonyl compounds – first and foremost, glyceraldehyde-3-phosphate and methylglyoxal. It has been suggested that various modifications of the enzyme – from the oxidation of the sulfhydryl groups of the active site to glycation with sugars – can lead to its inactivation, which causes a direct increase in glyceraldehyde-3-phosphate concentration and an indirect increase in the content of other aldehydes. This “primary inactivation” of glyceraldehyde-3-phosphate dehydrogenase promotes its glycation with aldehydes, including its own substrate, and a further irreversible decrease in its activity. Such a cycle can lead to numerous consequences – from the induction of apoptosis, which is activated by modified forms of the enzyme, to glycation of amyloidogenic proteins by glycolytic aldehydes. Of particular importance during the inhibition of glyceraldehyde-3-phosphate dehydrogenase is an increase in the content of the glycating compound methylglyoxal, which is much more active than reducing sugars (glucose, fructose, and others). In addition, methylglyoxal is formed by two pathways – in the cascade of reactions during glycation and from glycolytic aldehydes. The ability of methylglyoxal to glycate proteins makes it the main participant in this protein modification. We consider the effect of glycation on the pathological transformation of amyloidogenic proteins and peptides – β-amyloid peptide, α-synuclein, and prions. Our primary focus is on the glycation of monomeric forms of these proteins with methylglyoxal, although most works are dedicated to the analysis of the presence of “advanced glycation end products” in the already formed aggregates and fibrils of amyloid proteins. In our opinion, the modification of aggregates and fibrils is secondary in nature and does not play an important role in the development of neurodegenerative diseases. The glycation of amyloid proteins with carbonyl compounds can be one of the triggers of their transformation into toxic forms. The possible role of glycation of amyloidogenic proteins in the prevention of their modification by ubiquitin and the SUMO proteins due to a disruption of their degradation is separately considered.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

The E3 ubiquitin-ligase SEVEN IN ABSENTIA like 7 mono-ubiquitinates glyceraldehyde-3-phosphate dehydrogenase 1 isoform in vitro and is required for its nuclear localization in Arabidopsis thaliana

The E3 ubiquitin-protein ligases are associated to various processes such as cell cycle control and diverse developmental pathways. Arabidopsis thaliana SEVEN IN ABSENTIA like 7, which has ubiquitin ligase activity, is located in the nucleus and cytosol and is expressed at several stages in almost all plant tissues suggesting an important role in plant functions. However, the mechanism underlying the regulation of this protein is unknown. Since we found that the SEVEN IN ABSENTIA like 7 gene expression is altered in plants with impaired mitochondria, and in plants deficient in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 1, we decided to study the possible interactions between both proteins as potential partners in plant signaling functions. We found that SEVEN IN ABSENTIA like 7 is able to interact in vitro with glyceraldehyde-3-phosphate dehydrogenase and that the Lys231 residue of the last is essential for this function. Following the interaction, a concomitant increase in the glyceraldehyde-3-phosphate dehydrogenase catalytic activity was observed. However, when SEVEN IN ABSENTIA like 7 was supplemented with E1 and E2 proteins to form a complete E1–E2–E3 modifier complex, we observed the mono-ubiquitination of glyceraldehyde-3-phosphate dehydrogenase 1 at the Lys76 residue and a dramatic decrease of its catalytic activity. Moreover, we found that localization of glyceraldehyde-3-phosphate dehydrogenase 1 in the nucleus is dependent on the expression SEVEN IN ABSENTIA like 7. These observations suggest that the association of both proteins might result in different biological consequences in plants either through affecting the glycolytic flux or via cytoplasm-nucleus relocation.     

Participation of glyceraldehyde-3-phosphate dehydrogenase in the regulation of 2,3-diphosphoglycerate level in erythrocytes

Data are presented concerning the possible participation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in regulation of the glycolytic pathway and the level of 2,3-diphosphoglycerate in erythrocytes. Experimental support has been obtained for the hypothesis according to which a mild oxidation of GAPDH must result in acceleration of glycolysis and in decrease in the level of 2, 3-diphosphoglycerate due to the acyl phosphatase activity of the mildly oxidized enzyme. Incubation of erythrocytes in the presence of 1 mM hydrogen peroxide decreases 2,3-diphosphoglycerate concentration and causes accumulation of 3-phosphoglycerate. It is assumed that the acceleration of glycolysis in the presence of oxidative agents described previously by a number of authors could be attributed to the acyl phosphatase activity of GAPDH. A pH-dependent complexing of GAPDH and 3-phosphoglycerate kinase or 2, 3-diphosphoglycerate mutase is found to determine the fate of 1,3-diphosphoglycerate that serves as a substrate for the synthesis of 2,3-diphosphoglycerate as well as for the 3-phosphoglycerate kinase reaction in glycolysis. A withdrawal of the two-enzyme complexes from the erythrocyte lysates using Sepharose-bound anti-GAPDH antibodies prevents the pH-dependent accumulation of the metabolites. The role of GAPDH in the regulation of glycolysis and the level of 2,3-diphosphoglycerate in erythrocytes is discussed.     

Metabolic changes in the African fruit beetle,Pachnoda sinuata,during starvation

Specimens of the fruit beetle Pachnoda sinuata were starved for up to 30 days. The weight of the beetles declined consistently throughout the starvation period. Concentrations of carbohydrates and alanine in flight muscles, fat body and haemolymph decreased rapidly after onset of starvation, while the concentration of proline remained high. Whereas the lipid concentrations in the haemolymph did not change significantly upon starvation, the lipid content in flight muscles and fat body decreased significantly.Beetles that had been starved for 14 days responded to injection of Mem-CC, the endogenous neuropeptide from its corpora cardiaca, with hyperprolinaemia and a decrease in the alanine level, but no such effect was monitored after prolonged starvation of 28 days. Regardless of the period of starvation, Mem-CC injection could not cause hypertrehalosaemia or hyperlipaemia, although carbohydrates were increased in fed beetles after injection.Flight ability of beetles that had been starved for 15 or 30 days was apparently not impaired. During such periods, beetles used proline exclusively as fuel for flight as evidenced by the increase in the level of alanine in the haemolymph and decrease of the level of proline; the concentrations of carbohydrates and lipids remained unchanged.Activities of malic enzyme and alanine aminotransferase (enzymes involved in transamination in proline metabolism), glyceraldehyde-3-phosphate dehydrogenase (enzyme of glycolysis), 3-hydroxyacyl-CoA dehydrogenase (enzyme of beta-oxidation of fatty acids) and of malate dehydrogenase (enzyme of Krebs cycle) were measured in fat body and flight muscles. In flight muscle tissue the maximum activity of NAD(+)-dependent malic enzyme increased, while that of glyceraldehyde-3-phosphate dehydrogenase decreased during starvation, and malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and alanine aminotransferase were unchanged. In fat body tissue, activities of NADP(+)-dependent malic enzyme and 3-hydroxyacyl-CoA dehydrogenase increased during food deprivation and activities of glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and alanine aminotransferase remained unchanged.     

Effect of glycolysis inhibition on mitochondrial function in rat brain

Inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase enhances the neural vulnerability to excitotoxicity both in vivo and in vitro through an unknown mechanism possibly related to mitochondrial failure. However, as the effect of glycolysis inhibition on mitochondrial function in brain has not been studied, the aim of the present work was to evaluate the effect of glycolysis inhibition induced by iodoacetate on mitochondrial function and oxidative stress in brain. Mitochondria were isolated from brain cortex, striatum and cerebellum of rats treated systemically with iodoacetate (25 mg/kg/day for 3 days). Oxygen consumption, ATP synthesis, transmembrane potential, reactive oxygen species production, lipoperoxidation, glutathione levels, and aconitase activity were assessed. Oxygen consumption and aconitase activity decreased in the brain cortex and striatum, showing that glycolysis inhibition did not trigger severe mitochondrial impairment, but a slight mitochondrial malfunction and oxidative stress were present.     

Changes in glycolytic activity of Lactococcus lactis induced by low temperature

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30 degrees C increased approximately 2.5-fold following a shift from 30 to 10 degrees C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.     

INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN MAMMALIAN NERVE BY IODOACETIC ACID

Abstract— Cat sciatic nerves were exposed to iodoacetate for a period of 5–10 min and after washing out the iodoacetate, the enzymes, glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating); EC 1.2.1.12) and lactate dehydrogenase ( l -lactate: NAD oxidoreductase; EC 1.1.1.27) were extracted from the high-speed supernatant fraction of nerve homogenates. Concentrations of iodoacetate as low as 2.5 m m could completely block activity of glyceraldehyde-3-phosphate dehydrogenase but had no effect on lactate dehydrogenase. These findings are in accord with the classical concept shown earlier for muscle that iodoacetate blocks glycolysis by its action on glyceraldehyde-3-phosphate dehydrogenase. A complete block of activity of the enzyme was found after treatment with 2 to 5 m m -iodoacetate for a period of 10 min and such blocks were irreversible for at least 3 h. Glyceraldehyde-3-phosphate dehydrogenase activity was NAD specific, with NADP unable to substitute for NAD. The results are discussed in relation to the effect of iodoacetate in blocking glycolysis and in turn the fast axoplasmic transport of materials in mammalian nerve.