Showing papers on "Glycolysis published in 1980"

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Abstract: Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H2 and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H2 addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent Km, Vmax, and Q10 values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H2 production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.

The regulation of glycolysis and the pentose phosphate pathway

Abstract: Publisher Summary This chapter discusses the regulation of glycolysis and the pentose phosphate pathway. Glycolysis and the pentose phosphate pathway are the two main pathways of carbohydrate degradation in plants. Sucrose and starch are the principal sources of substrates for glycolysis in plants. The enzymes involved in the breakdown of sucrose and starch in plants do not appear to have regulatory properties. The localization of metabolic pathways in subcellular compartments is an aspect of control that has significance for the regulation of carbohydrate metabolism in plants. The chapter presents compartmentation of the enzymes glycolysis. Glycolysis occurs in plastids and in the cytoplasm of plant tissues; the reactions in the respective compartments are catalyzed by separate isoenzymes. The relative amounts of plastid and cytoplasmic isoenzymes differ for the various glycolytic reactions and depend on the type of tissue and its stage of development.

The Development of Enzymes of Energy Metabolism in the Brain of a Precocial (Guinea Pig) and Non‐Precocial (Rat) Species

Abstract: Key enzymes of ketone body metabolism (3-hydroxybutyrate dehydrogenase, 3-oxo-acid:CoA transferase, acetoacetyl-CoA thiolase) and glucose metabolism (hexokinase, lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase) have been measured in the brains of foetal, neonatal, and adult guinea pigs and compared to those in the brains of neonatal and adult rats. The activities of the guinea pig brain ketone-body-metabolising enzymes remain relatively low in activity throughout the foetal and neonatal periods, with only slight increases occurring at birth. This contrasts with the rat brain, where three- to fourfold increases in activity occur during the suckling period (0-21 days post partum), followed by a corresponding decrease in the adult. The activities of the hexokinase (mitochondrial and cytosolic), pyruvate dehydrogenase, lactate dehydrogenase, and citrate synthase of guinea pig brain show marked increases in the last 10-15 days before birth, so that at birth the guinea pig possesses activities of these enzymes similar to the adult state. This contrasts with the rat brain where these enzymes develop during the late suckling period (10-15 days after birth). The development of the enzymes of aerobic glycolytic metabolism correlate with the onset of neurological competence in the two species, the guinea pig being a "precocial" species born neurologically competent and the rat being a "non-precocial" species born neurologically immature. The results are discussed with respect to the enzymatic activities required for the energy metabolism of a fully developed, neurologically competent mammalian brain and its relative sensitivity to hypoxia.

Brain hexokinase, the prototype ambiquitous enzyme.

Abstract: Publisher Summary The brain is a voracious consumer of glucose—and of oxygen because the predominant route of glucose metabolism is aerobic oxidation via glycolysis and the tricarboxylic acid (TCA) cycle. Under special circumstances that lead to ketosis, ketone body metabolism might contribute to the generation of metabolic energy in the brain, but even under these conditions, an absolute requirement for a basal level of glucose metabolism remains. The critical dependence of normal brain function on an adequate supply of blood-borne glucose and oxygen has been demonstrated. Transport of glucose through the blood-brain barrier is a carrier-mediated process. The principal metabolic pathway involved is glycolysis. Under most conditions, regulation of glucose utilization equates with regulation of glycolysis. The principal control points for cerebral glycolysis are the hexokinase and phosphofructokinase reactions. Brain hexokinase offers a challenging opportunity for understanding how molecular structure and cellular structure might be inter-woven in regulating the in vivo activity of an enzyme of central importance for cerebral energy metabolism.

The swift increase in alcohol metabolism. Time course for the increase in hepatic oxygen uptake and the involvement of glycolysis

Abstract: Gastric intubation of female Sprague-Dawley rats with 5 g of ethanol/kg body wt. nearly doubled oxygen uptake by the isolated perfused rat liver maximally after only 2.5 h of treatment (Swift Increase in Alcohol Metabolism). Inhibition of enhanced oxygen uptake by KCN (2mM) and 4-methylpyrazole (0.8 mM) suggested the involvement of the mitochondrial respiratory chain and alcohol dehydrogenase in this phenomenon. Glycolysis was depressed after ethanol treatment. Diminished ATP generation via glycolysis accounts for a portion (23-50%) of the increased oxygen uptake, assuming that other rates of biosynthesis remain constant. Injection of adrenaline (2 mg/kg) 1 h before perfusion mimicked partially the action of ethanol on hepatic oxygen uptake. The increases produced by ethanol and adrenaline were not additive, suggesting that adrenaline is involved in the action of ethanol. Moreover, the increase in hepatic oxygen uptake produced by 2.5 h of ethanol treatment could be blocked by either alpha-(phenoxybenzamine; 40 mg/kg) or beta-(propranolol; 40 mg/kg) adrenergic blocking agents. Blood glucose increased after ethanol treatment, supporting the involvement of glycogenolytic hormones in this effect. These data indicate that at least part of the stimulated oxygen uptake after treatment with ethanol is a result of lower rates of glycolytic ATP generation resulting from hormone (e.g. adrenaline etc.) action. The ADP not phosphorylated in the cytosol enters the mitochondria, where it stimulates oxygen uptake.

Glycolysis in Trypanosoma brucei

Abstract: The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360--364] constitute a separate pool of glycolytic intermediates within the cell was investigated. In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell-sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities of the latter enzymes were all latent between 70 and 92%. Latency was abolished by addition of 0.1% Triton X-100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co-factors of glycolysis. The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes: (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol-3-phosphate, however, increased fourfold. (b) The phosphate potential was drastically lowered from 2900 to 450 M-1. (c) The trypanosomes became more reduced, as monitored by a change in the apparent redox state of the NADH/NAD+ courple from E'h = -189 to E'h = -219 mV. From the high levels of metabolite concentrations found and from comparison of the apparent mass-action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.

The decline in energetic metabolism with aging of the erythrocyte and its relationship to cell death

Abstract: Human erythrocytes were separated by buoyant density ultracentrifugation into fractions of progressively increasing mean cell age to measure the changes in glycolytic activity that occur during their 120-day life-span. The maximal activities of all glycolytic enzymes were shown to decline exponentially with cell age. Only three glycolytic enzymes exhibited a marked rate of decline with a t1/2 shorter than the cell life-span: hexokinase, aldolase, and pyruvate kinase. Glucose utilization, when measured in steady-state conditions (1 mM inorganic phosphate), showed a fourfold decrease through the erythrocyte life-span; lactate production also declined, but at a slower rate. When incubating conditions were altered by the introduction of a metabolic stimulus (either high phosphate for glycolysis, or methylene blue for the pentose pathway) the youngest cell fractions responded with decidedly increased rates of glucose consumption and lactate production. However, this ability gradually declined with cell aging, and ultimately, the oldest cells had metabolic rates as low as if there were no stimulus present. The oldest erythrocytes appear to have lost the flexibility needed to respond to metabolic stress and are more vulnerable to events in the circulation that may require the ability to increase the basal rate. This defect is probably responsible for the disappearance of aged erythrocytes from the circulation.

Starvation and the activities of glycolytic and gluconeogenic enzymes in skeletal muscles and liver of the plaice,Pleuronectes platessa

Abstract: Changes in body index parameters and liver, red muscle and white muscle enzyme profiles have been determined in fed and four month starved plaice,Pleuronectes platessa. The results are compared to other vertebrates to estimate specific tissue metabolic patterns and changes in these patterns with starvation. 1. Liver demonstrates the lowest glycolytic but highest gluconeogenic capacity of the three tissues. Red muscle has little, if any, gluconeogenic potential, based upon low activities of phosphoenol pyruvate carboxykinase and glucose-6-phosphatase and no detectable activities of pyruvate carboxylase. Plaice white skeletal muscle has the highest glycolytic potential of the tissues studied. 2. Plaice starved for four months demonstrate significant reductions in liver-somatic index and red muscle-somatic index, and increases in tissue water contents (Table 1). Enzyme activities generally decline in both muscle types, but are maintained in the liver (Table 2). Activities of liver soluble phosphoenol pyruvate carboxykinase increase by approximately 8-fold, suggesting that the enzymic response to starvation in plaice is similar to that of mammals. 3. These results suggest that starvation in plaice is associated with both a decrease in spontaneous activity and metabolic capacity of skeletal muscles, and an enhanced potential for liver gluconeogenesis. Also, it is possible that the precursors for liver gluconeogenesis do not form pyruvate as an intermediate step.

Lactate: A major product of glutamine metabolism by human diploid fibroblasts

Abstract: Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.

Distribution of enzymes of glycolysis and gluconeogenesis in fish tissues

Abstract: Gluconeogenesis in fishes has been demonstrated in whole animals and liver preparations. However, at present, the relative physiological importance of possible substrates such as lactate, pyruvate and amino-acids or the precise sites of gluconeogenesis are unclear. In mammals, gluconeogenesis takes place in the liver and kidney, and the same could occur in fishes although it has been proposed that fish red muscle is also capable of reconverting lactate (derived from white muscle) to glucose. In this present study, the activities of 3 key glycolytic (hexokinase, phosphofructokinase and pyruvate kinase) and 2 key gluconeogenic (fructose diphosphatase and phosphoenolpyruvate carboxykinase) enzymes were investigated in tissues of the rainbow trout Salmo gairdneri, the cod Gadus morhua, and the plaice Pleuronectes platessa in order to elucidate the relative glycolytic/gluconeogenic capacities of the individual fish tissues. The glycolytic enzymes were found in all tissues, the relative potential being skeletal muscle>heart, brain >kidney, gills>liver. The gluconeogenic enzymes were not present in all tissues, and were mainly concentrated in the liver and kidney. Hence the results indicate that the liver, and to a lesser degree, the kidney are the major sites of gluconeogenesis in fishes, and that the process is unlikely to occur in skeletal muscle.

Isolation of a Chinese hamster fibroblast mutant defective in hexose transport and aerobic glycolysis: its use to dissect the malignant phenotype

Abstract: A procedure is described for the selection of glucose uptake mutants based upon radiation suicide of Chinese hamster fibroblasts by 2-deoxy[3H]glucose. In one of these mutants, DS 7, the ability to transport either 2-deoxyglucose or 3-O-methylglucose was decreased to one-fifth to one-fourth. Besides this defect, DS7 produces 1/14th the lactic acid produced by the parent when grown on 5 mM glucose. This block in aerobic glycolysis is due to a mutation that affects the expression of the phosphoglucose isomerase gene because no isomerase activity is detected in cell extracts of DS7. This glycolytic block makes that cell line dependent exclusively on respiration for its energy requirement. Consequently, DS7 survives well after removal of glucose but dies quickly in the presence of oligomycin. The parental line O23 (subclone of CCl39) grows at low serum concentration, is anchorage-independent, and is tumorigenic in nude mice. The derived glycolytic mutant DS7 has retained both the in vitro transformed phenotype (low serum dependence and loss of anchorage dependence) and the tumor-forming capability. The tumor cells derived from the injection of DS7 cells have kept the original glycolytic defect. This finding suggests that the transformed properties (high hexose transport and aerobic glycolysis) that can be uncoupled from abnormal growth control are not necessary for the expression of the malignant phenotype in fibroblasts.

An activation factor of liver phosphofructokinase

Abstract: Pure phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from liver is strongly inhibited by ATP, whereas crude phosphofructokinase is only slightly inhibited by ATP. A factor that is removed from the enzyme during purification and can prevent the inhibition of phosphofructokinase by ATP has been isolated. The factor can be resolved into three components that differ in molecular weights, as shown by gel filtration on Sephadex G-25. These factors overcome the ATP inhibition but have no effect on the catalytic activity under the optimum assay conditions. Furthermore, AMP acts syngeristically with the activation factor in reversing ATP inhibition. It is proposed that the activation of phosphofructokinase by the activation factor and AMP is sufficient to account for the glycolytic flux in the liver.

Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications.

Abstract: 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.

Effects of Glucose on Insulin Release and on Intermediary Metabolism of Isolated Perifused Pancreatic Islets from Fed and Fasted Rats

Abstract: We examined the relationship between glucose-induced insulin release and the intermediary metabolism of islets from fed and fasted rats. Isolated islets were perifused and insulin release measured in the effluent. At various times after switching islets from 2.4 to 8.6 or 14.5 mM glucose or from 2.4 to 14.5 and back to 2.4 mM glucose, islets were quickly frozen, freeze dried, and subsequently analyzed for tissue content of glucose-6-P, fructose-1,6-P 2 plus triose-P, P 1 , ATP, ADP, 59-AMP, NADH, NADPH, total NAD, and total NADP using enzymatic fluorometric procedures. When islets from fed rats were exposed to high glucose, there were concomitant increases of insulin release and islet content of glucose-6-P, fructose-1,6-P 2 plus triose-P, NADH, and NADPH. During stimulation P 1 and 59-AMP content fell markedly. The total adenine nucleotide content remained constant. Similar secretory and metabolic changes occurred when 1.5 mM P 1 was added to the perifusion fluid. When glucose-stimulated islets were switched back to low glucose for 10 min, all substances but fructose-1,6-P 2 plus triose-P, 59-AMP, NADPH, and possibly ATP returned to the prestimulatory level. Starvation of rats for 3 days blocked the secretory response to 8.6 mM glucose. Fructose-1,6-P 2 plus triose-P rose but it did not attain the level existing in islets from fed rats. The ratios (ATP)/(59-AMP) and (ATP)/(P 1 ,)(ADP) increased to the values observed in glucose-stimulated islets of fed rats. The metabolic changes in islets from fed rats exposed to high glucose are consistent with an activation of glycolysis occurring concomitantly with stimulated rates of insulin release. This occurs despite the decrease of important activators of glycolysis—P 1 and 59-AMP. The enhanced glycolysis possibly results from P-fructokinase activation by increased fructose-6-P levels. Activation of glycolysis with 8.6 mM glucose was not as pronounced in islets from starved rats. Despite the different secretory response of islets from fed and fasted rats, the changes of phosphorylation state in the islets, in particular, P 1 , and 59-AMP levels, were similar.

Control of glycolysis in the posterior adductor muscle of the sea musselMytilus edulis

Abstract: Concentrations of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate, phosphoarginine and citrate have been estimated after various periods of valve closure (Table 1 and Fig. 1). Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals glycogen phosphorylase, phosphofructokinase, hexosediphosphatase and pyruvate kinase catalyze non-equilibrium reactions. The first three enzymes possess relatively low activities (Table 2).

Studies of the biochemistry of contracting and relaxing muscle by the use of 31P n.m.r. in conjunction with other techniques

Abstract: When n.m.r. is applied to suitably chosen biological problems it yields a wealth of fundamental information unmatched by any other technique. By means of 31 P n.m.r. we have studied intact living muscle at rest, during contraction and during recovery from contraction. Phosphocreatine, ATP, inorganic phosphate, phosphorylated intermediaries of glycolysis, pH and the binding of Mg 2+ to ATP are observed directly in the spectra. From the spectra can be calculated the concentration of free ADP, the free energy change for ATP hydrolysis, the production of lactic acid and the total ATP turnover. Changes in these quantities can thus be followed continuously in vivo and we have shown how they are related to the decline in force development and to the slowing of relaxation that occur during fatigue. Similar methods have been applied to study the control of glycolysis.

Mechanism of action of glucagon on hepatocyte phosphofructokinase activity.

Abstract: Addition of glucagon to isolated hepatocytes reduced the activity of 6-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40). Phosphorylation contributed to the inhibition of pyruvate kinase, but several lines of evidence indicated that this reaction was not responsible for the inhibition of phosphofructokinase. First, the increase in phosphorylation in intact cells induced by increasing the concentration of glucagon did not correlate well with the decrease in enzyme activity. Second, phosphorylation of phosphofructokinase induced by addition of cyclic AMP and Mg2+-ATP or by addition of Mg2+-ATP and the catalytic subunit of the cyclic AMP-dependent protein kinase to hepatocyte extracts had no effect on enzyme activity. Third, ammonium sulfate precipitation of the enzyme from extracts of cells incubated with glucagon abolished the hormone effect. The effect could be restored, however, by the addition of a phosphofructokinase-free extract from glucagon-treated cells to the ammonium sulfate-treated enzyme from either untreated or glucagon-treated cells. These results suggest that the inhibition of phosphofructokinase by glucagon is due to changes in the level of an allosteric effector(s).

Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes

Abstract: Intracellular enzymes in erythrocytes can be made accessible for in situ kinetic studies by treating the cells with bifunctional reagents to crosslink proteins, thus creating a network that allows subsequent permeabilization by delipidation without escape of intracellular proteins. Dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate, and toluene-2,4-diisocyanate have been used successfully as crosslinking reagents, and digitonin has been used for delipidation. In a systematic study of the in situ behavior of the 11 glycolytic enzymes of rat erythrocytes, it was observed that Km and Vmax values for the majority of the enzymes are essentially the same in situ as in vitro. Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) is inhibited by excess of pyruvate as much in situ as in vitro. Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was allosterically inhibited by glucose 6-phosphate nearly as much in situ as in vitro but was not affected by 2,3-biphosphoglycerate. The allosteric properties of 6-phosphofructokinase (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], and pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) in situ were qualitatively similar to those observed in vitro, but some important quantitative differences were noticed. Particularly striking was the much greater activity of phosphofructokinase in situ compared to that in vitro at physiological concentrations of effector metabolites.

Energy metabolism during reperfusion following ischemia

Abstract: Insulin treatment of hearts during aerobic reperfusion following transient ischemia in the working rat heart preparation significantly improved the recovery of myocardial function. This improvement was reflected both by a shorter time required for the heart to resume beating, and to increase heart rate and peak systolic pressure after resumption of beating. The beneficial effects of insulin may be related to improved energy metabolism secondary to small increases in pyruvate production during the early phase of reperfusion. It was also associated with an increased rate of restoration of cellular K+. Pyruvate addition to the perfusate also improved resumption of spontaneous beating of the heart and restoration of normal rate and pressure development. This effect of pyruvate was also associated with increased cellular levels of K+. Both insulin and pyruvate may improve ATP production during the first few minutes of reperfusion when glycolysis and oxidation of fatty acids are inhibited, but ATP levels were not increased after 30 min of reperfusion.

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—II. High energy phosphate compounds and the role of glycolytic and gluconeogenetic enzymes

Abstract: 1. 1. ATP, ADP, AMP, energy charge potential and total adenylates in heart, kidney and muscle are relatively unaffected by environmental hypoxia. In the liver, hypoxia causes a 90% drop in ATP, a rise in ADP and AMP, and a drop in energy charge potential and total adenylates. In the muscle tissue ATP concentration is stabilized by a large creatine phosphate pool. 2. 2. Hexokinase activity in the heart is 20 times higher than in the swimming muscle, and thus the heart has a high potential for utilizing exogenous glucose as an anaerobic substrate. 3. 3. The role of creatine phosphate in regulating muscle glycolysis is discussed on background of the strong inhibition of muscle phosphofructokinase by physiological concentrations of creatine phosphate. 4. 4. Flounder heart has a dominating M-type lactate dehydrogenase which is identical to the muscle enzyme by electrophoretic and kinetic criteria. This improves the anaerobic capabilities of the flounder heart compared to other fish hearts. 5. 5. Both liver and kidney have high activities of the gluconeogenetic enzymes glucose-6-phosphatase, fructose-1,6-diphosphatase, and phosphoenolpyruvate carboxykinase and both are capable of synthesizing glucose from [14C]lactate. Because of more favorable energy conditions in the kidney this organ may substitute the liver as a gluconeogenetic organ during hypoxia.

Relationship between increased aerobic glycolysis and DNA synthesis initiation studied using glycolytic mutant fibroblasts.

Abstract: Reports from several laboratories have suggested increased rates of glycolysis play an essential part in the initiation of DNA synthesis. This is based on observations that aerobic glycolysis: (1) occurs at low rate in resting mammalian cells and at very high rate in tumour cells; (2) increases rapidly after DNA synthesis is initiated by addition of serum or purified growth factors, and (3) correlates with the expression of the transformed phenotype. Also, specific inhibitors of aerobic glycolysis prevent the initiation of DNA synthesis. To determine whether the rapid activation of phosphofructokinase--and therefore glycolysis--by purified growth factors is necessary for the initiation of cell proliferation, we have isolated and studied two classes of glycolytic mutants. The first, isolated from Chinese hamster fibroblasts, has a total block in the glycolytic pathway. The second, from hamster and Fisher rat fibroblasts maintains a permanent high rate of glycolysis. We have found that both classes of mutants retain normal control of DNA synthesis in response to serum. This dissociation indicates that growth-factor-stimulated glycolysis is not involved in the control of initiation of DNA synthesis and cell proliferation.

Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

Abstract: Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 2.7.1.40) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11 +/- 0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 +/- 0.04 U/microgram DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +/- 0.13; hypoxia, 0.94 +/- 0.13 microgram enzyme protein/microgram DNA). Specific activity was not significantly different after 96 h (L2: normoxia, 261 +/- 11; hypoxia, 261 +/- 14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic O2 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to charcterizing enzymes, as well as quantitating cellular enzyme content.

Biochemical and histochemical alterations in skeletal muscle in man during a period of reduced activity

Abstract: This study investigated the hypothesis that the adaptations in skeletal muscle to prolonged exercise overload, involving high levels of adenosine triphosphate (ATP) resynthesis, result in a preferential adaptation to pathways involved in energy metabolism. The change in selected properties of skeletal muscle during a period of reduced activity was used as an indication of training-induced adaptations. Muscle biopsy samples from the vastus lateralis were analyzed 6 weeks and 18 weeks after a 5-month, intense, intermittent training program. Significant reductions occurred (p less than 0.05) in enzyme activities representative of the citric acid cycle (succinic dehydrogenase, SDH), beta oxidation of free fatty acids (3-hydroxyacyl CoA dehydrogenase, HADH), glycogenolysis (total phosphorylase, PHOSP), and glycolysis (phosphofructokinase, PFK). In addition, reductions in concentration (p less than 0.05) were also found for ATP, creatine phosphate (CP), and glycogen. With the exception of PFK, all enzyme changes and the high energy phosphates reached new stable levels by at least the 6th week of detraining. The absence of changes in muscle cell type and size during the detraining period supports the hypothesis that adaptations in energy potential of the muscle cell predominate in this type of high intensity overload situation.

Integration of Pathways of Synthesis and Degradation of Hexose Phosphates

Abstract: Publisher Summary This chapter discusses the integration of pathways of synthesis and degradation of hexose phosphates. In higher plants, hexose phosphates are formed in photosynthesis and gluconeogenesis, and also from the breakdown of storage carbohydrates; they are consumed by glycolysis and the oxidative pentose phosphate pathway, and also in the synthesis of oligo- and polysaccharides. Thus, hexose phosphates play a dominant role in carbohydrate metabolism and consequently, in the economy of the plant as a whole. The reductive and oxidative pentose phosphate pathways, glycolysis, and gluconeogenesis share other intermediates in addition to hexose phosphates. In the studies of the control of hexose phosphate metabolism, the evidence strongly indicates that most of the control is exercised by the selected regulatory enzymes. Such enzymes are regarded as those which respond to the original metabolic signal and which initiate changes in the activities of the remaining enzymes of the pathways.