The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

CONTROL OF AEROBIC GLYCOLYSIS IN GUINEA-PIG CEREBRAL CORTEX SLICES

—The effect of glutamate on aerobic glycolysis in guinea-pig cerebral cortex slices was analysed in comparison with that of high-potassium. In contrast to the increased glycolysis in 50 mm -potassium medium which was accompanied by increases of fructose diphosphate and triose phosphates in the slices, the addition of 5 mm -d -glutamate to the medium increased the rate of glycolysis without increasing these intermediates. When increasing the concentration of potassium in the medium up to 20 mm , the rate of aerobic glycolysis was not increased although fructose diphosphate and triose phosphates in the slices were increased. At this potassium concentration in the medium ATP in the slices was highest. At 30 mm -potassium the rate of glycolysis was increased significantly, but fructose diphosphate and triose phosphates were decreased. ATP was lower at 30 mm - than at 20 mm -potassium. By increasing potassium to 40 mm and above, the rate of glycolysis was further increased, and fructose diphosphate and triose phosphates were again increased. Between 5 and 20 mm -potassium in the medium the increasing effect of glutamate on glycolysis was very pronounced. d -Glutamate decreased the amounts of ATP, fructose diphosphate and triose phosphates at any concentration of potassium in the medium. When adding cyclic AMP and 5′AMP to the slices, fructose diphosphate and triose phosphates were increased, but the rate of glycolysis was not increased. On the basis of these observations mechanisms of the control over glycolysis in guinea-pig cerebral cortex slices are discussed. It is suggested that the glycolysis is controlled by the changes in ATP concentration through their action on the glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase system. The changed patterns of the glycolytic intermediate profile in the slices when adding ATP to the medium are consistent with this suggestion. The addition of l -phenylalanine to guinea-pig cerebral cortex slices did not inhibit the rate of glycolysis, although it inhibited the activity of pyruvate kinase.     

Control of glycolysis by orthophosphate and adenosine triphosphate in soluble extracts of germinating pea seeds

Summary Control of aerobic glycolysis by adenosine triphosphate and orthophosphate has been studied in cell-free extracts of germinating pea seeds. Orthophosphate accelerates glycolysis under all conditions studied. At high concentrations of magnesium ion ATP accelerates glycolysis, whereas at lower magnesium concentrations ATP severely inhibits glycolysis. The inhibitory effect of ATP is markedly relieved by orthophosphate. Metabolite analyses suggest an important regulatory role of phosphofructokinase and show that low ratios of F-6-P: FDP accompany the appearance of a high rate of glycolysis, and vice versa. Thus, ATP raises the F-6-P: FDP ratio at low magnesium levels, while P_i lowers this ratio. At high Mg²⁺ (where ATP accelerates glycolysis), ATP causes a low F-6-P: FDP ratio to appear. At low Mg²⁺ concentration, orthophosphate accelerates glycolysis by activation of phosphofructokinase; at high magnesium concentration, the chief effect of orthophosphate is its long-known role in facilitating the oxidation of triose phosphate.     

Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.     

THE EFFECT OF CARBON DIOXIDE TENSION ON TISSUE METABOLISM (RETINA)

The metabolism of rat retina was found to be sensitive to the concentration of the carbon dioxide-bicarbonate buffer system. Increasing the carbon dioxide from 1 per cent to 5 per cent at constant pH nearly doubled both respiration and glycolysis. Increasing the carbon dioxide at constant pH from 5 per cent to 20 per cent had no effect on glycolysis, but depressed the Q _OO2 from 31 to 19. In a medium containing glucose and the 1 per cent carbon dioxide-bicarbonate buffer, the addition of succinate increased the Q _OO2 from 12 to 26, without affecting glycolysis. In a medium containing glucose and phosphate, succinate had no significant effect.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

Effect of salts on the activity of carrot phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) from carrot roots was activated by a number of salts. Increase in salt concentration beyond the optimum generally led to a decrease in enzyme activity. Salts of the multivalent anions sulfate and phosphate were very effective activators and inhibitors. Potassium acetate and potassium succinate were also activators. Potassium tartrate and potassium citrate produced a small stimulation at low concentration but with further increase they became inhibitory. The results suggested that the salt effect was largely due to anions rather than cations. Salts such as NaCl, KCl, and in particular potassium phosphate, relieved the inhibition of carrot phosphofructokinase by phosphoenolpyruvate. KCl and potassium phosphate also reversed the inhibition of carrot phosphofructokinase by citrate. The possible significance of these observations in the regulation of glycolysis and carbohydrate metabolism, and in salt respiration is discussed.     

Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Vanadate activates pentose phosphate pathway and glycolysis, and raises fructose 2,6-bisphosphate concentration in slices of lactating rat mammary gland.

In mammary gland slices from lactating rats, vanadate increased the rate of glucose oxidation via the pentose phosphate pathway by 36% and raised the glucose flux via glycolysis by 47%. Furthermore, vanadate increased the fructose 2,6-bisphosphate (Fru-2,6-P2) level by 33%. The effect of vanadate on glucose oxidation was compared to the effect of insulin. The present data indicate that 0.5mM vanadate has an effect on glucose utilization similar to that of insulin but does not reach the same level.     

THE EFFECT OF LOW OXYGEN TENSION ON TISSUE METABOLISM (RETINA)

Lactic acid production by rat retina in a medium containing phosphate was studied chemically. One half as much lactic acid was found as in a medium containing bicarbonate. In our experience the rate of respiration in a phosphate medium was sensitive to oxygen tension, for it was 38 per cent lower at 10 per cent and 51 per cent lower at 5 per cent oxygen than at 100 per cent oxygen. Previously Laser had reported no decrease in respiration at 5 per cent oxygen in phosphate medium. In phosphate medium, when the oxygen tension was varied, respiration and glycolysis bore a reciprocal relationship to each other. In bicarbonate medium, when the oxygen tension was lowered from 95 per cent to 5 per cent there was no significant change in the respiration, but glycolysis was increased nearly to the anaerobic level. This agrees with the earlier experiment of Laser in bicarbonate medium and adds support to his conclusion that the rate of glycolysis is controlled by oxygen tension rather than by the rate of respiration, under the conditions of the experiment.     

Catalytic properties of alpha-ketoglutarate decarboxylase from bovine brain]

Investigation of the effect of different buffer systems on the rate of alpha-ketoglutarate decarboxylase reaction have shown that the pH optimum is 6.8 in tris-maleic, tris-H3PO4 and KH2PO4-KOH buffers, and it is 7.5 in imidazole buffer. The highest reaction rate was observed when using phosphate containing buffers. The increase of phosphate concentration increased considerably the rate of alpha-ketoglutarate decarboxylase reaction. Mg2+ and Ca2+ were shown to affect slightly the reaction rate. Co2+ and Ag+ slightly inactivated the enzyme. Cu2+ turned to be a very efficient inhibitor of alpha-ketoglutarate decarboxylase reaction. Apparent Mikhaelis constants are determined to be 1.6-10(-3) M for alpha-ketoglutaric acid and 1.7-10(-2)M for 2,6-dichlorphenolindophenol.     

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg²⁺.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group.     

Phorbol esters, bombesin and insulin elicit differential responses on the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase system in primary cultures of foetal and adult rat hepatocytes.

The effects of 4 beta-phorbol 12-myristate 13-acetate (PMA), bombesin and insulin on 6-phosphofructo-2-kinase (PFK-2) activity, on fructose 2,6-bisphosphate concentration and on the phosphorylation state of PFK-2 were investigated in primary cultures of hepatocytes from foetal and adult rats. Bombesin stimulated PFK-2 activity and increased hexose phosphate (glucose 6-phosphate and fructose 6-phosphate) and fructose 2,6-bisphosphate content in hepatocytes both in the foetal and adult state. However, PMA-treated foetal cells exhibited a marked stimulation in fructose 2,6-bisphosphate concentration and in PFK-2 activity as well as in the content of hexose phosphates, while no response was found in the case of adult hepatocytes. Moreover, the effect of PMA on foetal hepatocytes was suppressed when cells were incubated with cycloheximide, but not when this effect was elicited by bombesin or insulin. These results, and those obtained on the phosphorylation state of PFK-2, suggest that there are different pathways that modulate fructose 2,6-bisphosphate content and, therefore, the control mechanisms of glycolysis and gluconeogenesis at this regulatory step, both in adult and foetal rat liver.     

Inhibition of tumor cell growth by a specific 6-phosphofructo-2-kinase inhibitor, N-bromoacetylethanolamine phosphate, and its analogues

The high rate of glycolysis despite the presence of oxygen and mitochondria in tumor cells implies an important role for this process in cell division. The rate of glycolysis is assumed to be dependent on the cellular concentration of fructose 2,6-bisphosphate, the concentration of which in turn depends on a bifunctional enzyme and the ratio of this enzyme's 6-phosphofructo-2-kinase versus its fructose 2,6-bisphosphatase activities. To prove the hypothesis that inhibition of glycolysis in tumor cells by 6-phosphofructo-2-kinase inhibitors would cause inhibition of tumor cell proliferation, ten N-bromoacetylethanolamine phosphate analogues were designed, synthesized, and tested. They were screened for their activities against various human tumor cell lines to study the effects of inhibition of glycolysis on cell proliferation. The relationship between the structure of these compounds and their inhibitory activity on cell proliferation was also discussed. It was found that the activity of N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide, and N-(3-methoxypropyl)-bromoacetamide was comparable to that of the positive control AraC. These three inhibitors showed in vivo anticancer effects in P388 transplant BDF1 mice.     

Accelerating effect of 2,4,6-trinitrophenol on the glycolytic rate of human red cells

A number of instantaneous changes occurred when picrate was added to a suspension of human red cells in steady state with respect to glycolysis and ion distribution across the membrane at pH 7.40. The rate of glycolysis increased, without change in glycolytic quotient, to a new steady-state value, the effect reaching a maximum of 1.75 times the rate of the control at 0.5 mM picrate. Inorganic phosphate (P(i)) was released at a relatively constant rate, increasing with picrate concentration to 1.0 mmol P(i)/liter cells x h at 5-6 mM picrate. The steady- state concentrations of ATP and 1,3-diphosphoglycerate (1,3-DPG) decreased to new stable values within 15-45 min after the addition of picrate. The ATP level was affected only at picrate concentrations of 1 mM or more, and the level of ATP stabilized at 75 percent of the control values at 4 mM of picrate. In contrast, 1,3-DPG concentrations decreased to 40 percent of the control value of 0.5 mM picrate. Higher concentrations of picrate resulted in only a small additional decrease in the stationary concentration of 1,3-DGP. A net efflux of cellular potassium at constant rate took place. This net efflux was an almost linear function of picrate concentration in the range of 0.1-3 mM. At the latter concentration the net efflux amounted to about 2.7 meq/liter cells x h and a further increase in picrate concentration caused only a minor increase in the potassium efflux. Possible mechanisms for the effects of picrate on human red cell glycolysis are discussed.     

Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes : II. Relative importance of glycolysis and oxidative phosphorylation on phytohaemagglutinin stimulation

The effect of inhibition of the oxidative phosphorylation of human blood lymphocytes in the presence and absence of phytohaemagglutinin has been investigated. It was found that the incorporation of inorganic phosphate into acid-soluble nucleotides is dependent on, though not a direct measure of oxidative phosphorylation. Optimal concentration for inhibition of oxidative phosphorylation with oligomycin and the uncoupler 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) were determined. Under conditions of maximally inhibited P_i incorporation into acid-soluble nucleotides (80%) and maximally increased oxygen consumption and lactate production (4–5 times), the stimulatory effect of phytohaemagglutinin on several glycolytic parameters could still be observed. Therefore, stimulation of cellular processes by PHA is still possible when energy is provided by glycolysis only.     

The relation between phosphate potential and growth rate of Streptococcus cremoris

During energy limited growth the phosphate potential of the adenine nucleotide pool of Streptococcus cremoris was independent of growth rate. The ratio of the phosphate potential and proton motive force under these conditions was 2.6 to 2.7. Under carbon-limiting conditions the phosphate potential increased with the growth rate which is the result of a decrease of the organic phosphate content of the cells at higher growth rates. The ATP/ADP ratio of energy- and carbon-limited cultures had the same value and it is proposed that this ratio is kept constant by a near equilibrium reaction in the glycolysis.     

Glucose transporters and transport kinetics in retinoic acid-differentiated T47D human breast cancer cells

The rates of glucose transport and of glycolysis and the expression of the glucose transporters GLUT-1 through GLUT-4 were measured in T47D human breast cancer cells that underwent differentiation by retinoic acid. Glucose transport was found to be the rate-limiting step of glycolysis in control and differentiated cells. The transporters GLUT-1, GLUT-3, and GLUT-4 were present in the cell membrane and in the cytoplasm, and GLUT-2 was present solely in the cytoplasm. Differentiation led to a reduction in GLUT-1 and to an increase in cytoplasmic GLUT-2 and GLUT-3 with no change in GLUT-4. Differentiation also caused a reduction in the maximal velocity of glucose transport by approximately 40% without affecting the Michaelis-Menten constant of glucose transport. These changes did not alter the steady-state concentration of the phosphate metabolites regulating cell energetics but increased the content of phospholipid breakdown phosphodiesters. In conclusion, differentiation of human breast cancer cells appears to be associated with decreased glycolysis by a mechanism that involves a reduction in GLUT-1 and a slowdown of glucose transport.     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

Suppression of the accumulation of triosephosphates and increased formation of methylglyoxal in human red blood cells during hyperglycaemia by thiamine in vitro

The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications. A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate. To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions. In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal. The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate. Biochemical changes implicated in the development of diabetic complications were thereby prevented. This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.