Showing papers on "Glycolysis published in 1971"

Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

Abstract: 1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (;working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [(14)C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and (14)CO(2)). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue contents of hexose phosphates and of citrate. There were similar findings when working hearts from streptozotocin-diabetic rats with insulin added to the medium were compared with normal hearts. 8. The effects of insulin addition or of the chronic diabetic state could be explained in terms of an action of insulin on glucose transport. Increased heart work also acted at this site, but in addition there was evidence for altered regulation of glycolysis mediated by changes in tissue contents of adenine nucleotides or of citrate.

Metabolic alterations in the human erythrocyte produced by increases in glucose concentration. The role of the polyol pathway.

Abstract: Human erythrocytes incubated in medium containing 50 mM glucose have increased intracellular sorbitol and fructose concentrations as compared with samples incubated with 5 mM glucose. Increased medium glucose concentration did not significantly alter total glucose consumption or lactate production. However, the intracellular lactate:pyruvate ratio rose, the concentrations of fructose diphosphate, and triose phosphates increased, and the 2,3-diphosphoglycerate concentration fell. [14C]O2 production from glucose-1-14C also increased with increased medium glucose concentration. These changes are believed to reflect changes in the redox states of the diphosphopyridine nucleotide/reduced form of diphosphopyridine nucleotide (NAD/NADH) and nicotinamide—adenine dinucleotide phosphate/reduced form of nicotinamide—adenine dinucleotide phosphate (NADP/NADPH) couples resulting from increased activity of the polyol pathway. Addition of pyruvate to the incubation media prevented these changes. These studies illustrate that an increase in the red cell's normal substrate, glucose, can produce changes in red cell metabolism.

Lactate production in the perfused rat liver

Abstract: 1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.

Selective reticulocyte destruction in erythrocyte pyruvate kinase deficiency

Abstract: Radioisotope studies of bilirubin turnover, ferrokinetics, and red cell survival ((51)Cr) in a patient with erythrocyte PK deficiency have provided evidence for prompt reticulocyte sequestration and destruction by the reticuloendothelial system. More mature erythrocytes appeared to survive well despite their deficiency of PK. PK-deficient reticulocytes, dependent upon oxidative phosphorylation for ATP production, are exquisitely sensitive to cyanide- or nitrogen-induced mitochondrial inhibition. If oxidative phosphorylation is unavailable, ATP levels decline rapidly, producing alterations in the cell membrane which allow massive losses of potassium and water. The result is a shrunken, spiculated, viscous cell whose rheologic properties would favor its sequestration by the reticuloendothelial system. Those reticulocytes with particularly low levels of PK exhibit very low glycolytic rates and thus are uniquely reliant upon oxidative phosphorylation. Other reticulocytes, better endowed with PK activity, can meet the increased ATP requirements of young erythrocytes. Upon reaching maturity, such cells have diminished ATP needs and can, therefore, survive despite their enzyme deficiency.

The Regulation of Pyruvate Kinase

Abstract: Publisher Summary This chapter discusses the molecular and kinetic properties, and the physiological significance of pyruvate kinase. From a thermodynamic consideration, these enzymes are especially suited for a control of glycolysis, because they catalyze unidirectional steps driving the metabolic flux into one direction. The combined action of the key enzymes of glycolysis and gluconeogenesis at the control points would allow futile cycles, resulting in the hydrolysis of energy-rich phosphate—an energy-wasting process. Additional regulatory properties of the key enzymes of glycolysis should therefore be expected in tissues that are able to carry out both glycolysis and gluconeogenesis, allowing an opposing control of both metabolic processes. Changes of pH in physiological range would, in addition, allow an opposing control of gluconeogenesis and glycolysis at the level of pyruvate kinase and pyruvate carboxylase reactions. Changes of the pH would not only control pyruvate kinase at suboptimal concentrations of PEP and antagonize the negative control of pyruvate kinase by ATP and alanine, but also control in an opposing way, at suboptimal levels of acetyl-CoA pyruvate carboxylase, the enzyme acting in direction of PEP-synthesis. This relationship adds strong support to a physiological significance of both the intracellular pH and acetyl-CoA in the control of glycolysis and gluconeogenesis in liver.

Metabolism of hepatomas of different growth rates in situ and during ischemia.

Abstract: In freeze-clamped rat liver and in hepatomas 9618-A and 5123-D (slower-growing) and 3924-A (rapidly growing), the initial concentrations and time course of changes during ischemia of adenine nucleotides, glucose, lactate, pyruvate, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, acetyl-CoA, acyl-CoA, glycerides, phospholipid, and total lipid were measured. Initial samples of the tissues were taken under anesthesia within 2 sec after severance of blood vessels, and subsequent samples were obtained during the ensuing ischemia up to 10 min. In hepatomas 5123-D and 3924-A, the initial concentrations of ATP, total adenine nucleotides, and the ATP/ADP ratio were markedly lower than those in livers. During ischemia in liver and in 5123-D and 9618-A, the concentrations of ATP, total adenine nucleotides, and the ATP/ADP ratio decreased while AMP levels increased. In the rapidly growing hepatoma, ischemia did not change ATP or AMP concentrations. The glucose and pyruvate concentrations were lower in all hepatomas than in liver. During ischemia, glucose levels increased in livers and in slow-growing hepatoma 9618-A but were not appreciably altered in the 2 other hepatomas. Initial lactate and glutamate concentrations were much higher in the rapidly growing hepatoma than in liver or in slower growing tumors; the concentration of 2-oxoglutarate was low in all tumors. The ratios of the initial concentrations of lactate/pyruvate and glutamate/2-oxoglutarate increased in hepatomas in parallel with growth rate. Acetyl-CoA, acyl-CoA, total glycerides, phospholipids, and total lipids in 3924-A were 20 to 50% of values observed in liver. In ischemia, the concentrations of acyl-CoA increased in liver but did not change in the hepatoma; the acetyl-CoA, glyceride, phospholipid, and total lipid content did not change either in liver or in 3924-A. The initial concentration of ketone bodies in fed rats was in the same range in the slower-growing tumors as in liver; however, it was low in the rapidly growing tumor. In starvation, the total ketone and 3-hydroxybutyrate concentrations increased over 10-fold in all tissues. Acetoacetate in livers increased 5- to 6-fold during starvation and 13- to 20-fold in the hepatomas. The results also indicate that there are a number of biochemical parameters, including initial lactate level, ability to maintain ATP, and the state of the cytoplasmic and mitochondrial oxidation-reduction couples, that exhibit a correlation with tumor growth rate.

Gluconeogenesis in the castor bean endosperm: I. Changes in glycolytic intermediates.

Abstract: The control points of the Embden-Meyerhof-Parnas pathway in germinating castor bean (Ricinus communis) endosperms are sought in two ways: (a) by measuring the amounts of various glycolytic intermediates at intervals during the germination; (b) by determining the crossover points appearing during anoxia.A significant departure from thermodynamic equilibrium between substrates and products is found at the level of fructose 1,6-diphosphatase and phosphofructokinase. A definite shift of this ratio is observed at the onset of active gluconeogenesis. The concentrations of phosphoenolpyruvate and 3-phosphoglyceric acid increase at the same time. Another departure from the expected equilibrium is also observed at the level of the pyruvate kinase.The imposition of anoxia on 5-day-old endosperms reveals two crossover points, at the level of the same enzymes. It is therefore concluded that they regulate the glycolytic flow.The maximal glycolytic flow, however, is only 1/10 of the gluconeogenic one. To account for this high gluconeogenic efficiency, it is postulated that gluconeogenesis and glycolysis occur in separate intracellular regions. The consistent departure from equilibrium between adenylates observed during the early stages of anoxia supports the concepts that the pools of glycolytic and gluconeogenic intermediates are indeed compartmented and that the two processes are independently regulated.

Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

Abstract: 1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP2−. Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP2− was the substrate varied. The Ki for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and βγ-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP2− was varied. AMP inhibition was competitive when MgATP2− was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.

Energy Metabolism in Human Erythrocytes: I. EFFECTS OF SODIUM FLUORIDE

Abstract: Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water. Of the other known sites of ATP utilization, membrane-bound ATPase is inhibited by fluoride, but the incorporation of fatty acids into membrane phospholipids is unaffected until ATP is depleted. The addition of methylene blue to fluoride-treated red cells regenerates NAD, permitting triose oxidation and the generation of 3-phosphoglycerate and 2,3-diphosphoglycerate. Enolase inhibition is then partially overcome by mass action, and sufficient glycolysis proceeds to maintain the concentration of ATP. This in turn prevents the massive cation and water loss, and permits membrane phospholipid renewal to proceed. Membrane ATPase activity is not restored by the oxidant so that normal cation leakage remains unopposed by cation pumping in red cells exposed to the combination of fluoride and methylene blue.

Energy cost of gluconeogenesis in rat liver

Abstract: The notion is presented that recycling of carbon between the steps pyruvate to P-enolpyruvate, and fructose diphosphate to fructose-6-P, is involved in the overall control of hepatic gluconeogenesis in both normal and pathological states. This results in a higher than theoretical energy cost for gluconeogenesis, which may be revealed by low apparent P:O ratios when calculated on the basis of the theoretical increment of 6 moles of ATP used per mole of glucose formed, compared with the observed increment of respiration when the rate of gluconeogenesis is altered. The concept is developed that the rate of gluconeogenesis is controlled either directly by changes in the concentration of factors modifying pyruvate carboxylase or indirectly by alterations of the activity of pyruvate kinase. If the activity of the latter enzyme is increased, flux through the loop Pyr → OAA → PEP → Pyr will be increased and net gluconeogenesis decreased. Since the inhibitory effects of alanine and ATP on pyruvate kinase can be overcome by low concentrations of fructose diphosphate, interactions in the segment of the gluconeogenic pathway from P-enolpyruvate to glucose, although not able to affect gluconeogenesis flux directly, can control it indirectly by alterations of the fructose diphosphate concentration. Interactions discussed are variations of the citrate concentration, which will affect the activity of phosphofructokinase, and altered metabolic states caused by changes of the state of reduction of the cytosolic pyridine nucleotides. By taking into account the possibility that high rates of carbon recycling between pyruvate and P-enol-pyruvate occur relative to the net rate of gluconeogenesis, the relationship between changes of the cytosolic pyridine nucleotide oxidation-reduction state and the overall control of gluconeogenesis is clarified.

2,3-diphosphoglycerate affects enzymes of glucose metabolism in red blood cells.

Abstract: THE bulk of organic phosphate in the erythrocyte exists in the form of 2,3-diphosphoglycerate (2,3-DPG), which is known to determine the position of the oxygen dissociation curve1,2 as well as to influence the glycolytic pathway as a cofactor of the monophosphoglyceromutase (MPGM) reaction3 and as an inhibitor of its own formation in the diphosphoglyceromutase reaction4. But whether 2,3-DPG inhibits the hexokinase reaction is unclear and reports are contradictory5–7.

Congenital Hemolytic Anemia in the Basenji Dog Due to Erythrocyte Pyruvate Kinase Deficiency

Abstract: Congenital hemolytic anemia in the Basenji dog resembles pyruvate kinase (PK) deficiency in man as it is characterized by an abbreviated erythrocyte life span, an intense reticulocytosis, type II autohemolysis and splenomegaly. Glucose utilization and lactate production were inadequate with respect to the immature cell population. Analysis of enzymes involved in erythrocyte glycolysis revealed a deficiency of pyruvate kinase.

Carbohydrate metabolism in the domestic fowl

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Activities of some enzymes involved in lipogenesis, gluconeogenesis, glycolysis and glycogen metabolism in chicks (Gallus domesticus) from day of hatch to adulthood☆

Abstract: 1. 1. Activities of some enzymes involved in lipogenesis, gluconeogenesis, glycolysis and glycogen metabolism were studied in the chick ( Gallus domesticus ) from day 1 to adulthood. Malic enzyme and glucose-6-phosphate dehydrogenase were negligible at day 1 and increased significantly during the first week and the declined, G-6-PDH showed great variability. 2. 2. Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were high at day 1 and then decreased. Fructose diphosphatase increased significantly during the first 2 weeks and remained high. 3. 3. Glutamic oxaloacetic transminase and glutamic pyruvic transaminase were also high at day 1 and then decreased. Both GOT and phosphoenolpyruvate carboxykinase showed a rise again at 14 weeks, possibly indicating a second phase of gluconeogenesis. 4. 4. Phosphorylase and phosphoglucomutase increased during the first week and then dropped, whereas liver glycogen stayed high for 3 weeks before it started to drop. Thus possibly phosphorylase is increased before glycogenolysis starts.

Glycolytic and pentose phosphate pathway enzymes in jejunal mucosa. Adaptive responses to alloxan-diabetes and fasting in the rat.

Abstract: Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, hexokinase, and pyruvate kinase activities were measured in the 105,000 × g supernatant fraction of jejunal mucosa and liver of control, alloxandiabetic, and 72-h fasted rats. Alloxan-diabetes was associated with elevated activities of these enzymes in jejunal mucosa. In contrast, fasting reduced the activities of these enzymes. Hepatic enzyme activities were reduced by both diabetes and fasting. Pair-feeding or a 24-h fast reduced the increased mucosal weight of diabetic rats to that of control animals, but jejunal enzyme activity remained similar to control values suggesting that hyperphagia and mucosal hypertrophy are not the sole cause of the elevated enzyme activity in diabetic jejunum. These studies demonstrate a unique response of jejunal mucosal glycolytic and NADPH-generating enzymes in alloxan-diabetes. The elevated activities of these enzymes in diabetic jejunal mucosa contrasts with the reductions in the activit...