Glycolysis in human erythrocytes containing elevated concentrations of 2,3-P2-glycerate

In studies on the mechanism of the inhibitory effect of 2,3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained:1) Glucose consumption and lactate production are reduced by 70 and 40% relative to normal erythrocytes in red blood cells containing five times the normal amount of 2,3,-P₂-glycerate (“high-diphosphoglycerate” cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the “high-diphosphoglycerate” cells.2) About 50% of the inhibition of glycolysis in “high-diphosphoglycerate” cells can be accounted for by the 2,3-P₂-glycerate-induced decrease of the red-cell pH. This fall of the red-cell pH which occurs as a consequence of the Donnan effect of the non-penetrating 2,3-P₂-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinvse.3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2,3-P₂-glycerate of glycolytic enzymes. From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2,3-P₂-glycerate concentration. A marked increase of 3-P-glycerate concentration in “high-diphosphoglycerate” cells suggests that also the enzyme pyruvate kinase is inhibited by 2,3-P₂-glycerate.4) The dependencies of net-change of 2,3-P₂-glycerate concentration on the red-cell pH are identical in normal and “high-diphosphoglycerate” cells indicating that the balance between formation and decomposition of 2,3-P₂-glycerate is the same in erythrocytes with normal and very high concentrations 2,3-P₂-glycerate.     

Effects of potassium antimony tartrate on rat erythrocyte phosphofructokinase activity

In an earlier study, we observed a marked accumulation of antimony in erythrocytes of rats administered potassium antimony tartrate (Sb) in drinking water. This observation has raised concerns of possible adverse effects on the hematological systems. A study was therefore carried out to investigate the effects of Sb on phosphofructokinase (PFK), a rate-limiting enzyme of erythrocyte glycolysis. Preincubation of PFK with Sb caused a marked inhibition of the enzyme with 95% loss of activity at 5 mM. In comparison, 5 mM sodium arsenite, a known enzyme inhibitor, reduced PFK activity by only 38%. Increasing the concentrations of fructose-6-phosphate (F6P) or magnesium had no effects on the inhibitory potency of Sb. Varying the concentrations of ATP and Sb produced a complex effect on PFK activity. At 1 mM ATP, 0.2 mM Sb was required for 50% inhibition (IC₅₀) of PFK but only 0.05 mM Sb was required for the same inhibition when the concentration of ATP was reduced to 0.2 mM. Glutathione (2–10 mM) and hemoglobin (8–40 <μ > M) partially protected the enzyme from the Sb effect, with the protection being more effective at low antimony concentrations. When Sb was added to assay mixtures after initiation of a PFK reaction with physiological concentrations of ATP (0.2 mM) and F6P (0.1 mM), PFK activity was approximately 50% inhibited by 0.5 mM Sb and completely inhibited by 5 mM Sb. In contrast, glucose utilization in whole blood was only 16% lower over an 8 hour incubation period in the presence of 5 mM Sb. It is concluded that while PFK is markedly inhibited by Sb under in vitro assay conditions, glycolysis in erythrocytes is not significantly affected except at very high Sb concentrations. The weak effect of Sb on glycolysis in erythrocytes may be due in part to the protective effect of hemoglobin and, to a lesser extent, glutathione on PFK. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 227–233, 1998     

Estimation of the fructose diphosphatase-phosphofructokinase substrate cycle in the flight muscle of Bombus affinis

1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the ³H/¹⁴C ratio in glucose 6-phosphate as well as the rate of ³H release to water after the metabolism of [5-³H,U-¹⁴C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4μmol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5°C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4μmol/min per g fresh wt.) and glycolysis was decreased (5.8μmol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27°, 21° and 5°C the rates of substrate cycling were 0, 0.48 and 10.4μmol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca²⁺-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca²⁺ concentration associated with the contractile process.     

Involvement of glycolytic metabolism in developmental inhibition of rat two-cell embryos by phosphate

To elucidate the mechanism by which phosphate induces developmental inhibition of rat 2-cell embryos, we examined the mutual effects of glucose and other glycolytic and non-glycolytic sugars, the non-metabolizable glucose analogue, and glycolytic inhibitors on the inhibitory effect of phosphate. In the absence of glucose, 30-49% of embryos treated with 10-500 microM phosphate were able to develop to morula and blastocysts. On the other hand, in the presence of 5 mM glucose, 10 microM phosphate decreased the developmental rate of 2-cell embryos to the 4-cell stage and completely inhibited the development beyond the 4-cell stage. In contrast, glucose showed no influence on development in phosphate-free medium. Similarly to glucose, the other glycolytic sugars fructose (5 mM) and mannose (5 mM) enhanced the inhibitory effect of 10 microM phosphate but had no influence in the absence of phosphate. In contrast, the non-glycolytic sugar and non-metabolizable glucose analogue N-acetylglucosamine and 3-O-methylglucose (3-O-MGlc), respectively, did not enhance the effects of phosphate. 2-Deoxyglucose (2DGlc), another glucose analogue that is non-metabolizable but is converted by hexokinase to 2DGlc 6-phosphate, at concentrations as low as 0.1 mM completely inhibited cell cycle progression of 2-cell embryos cultured in glucose-free (Glc(-)) medium with 10 microM phosphate. In contrast, in the absence of phosphate, 2DGlc at the same concentration allowed 55% of 2-cell embryos to develop to morula and blastocyst stages. Addition of an inhibitor of enolase in glycolysis, sodium fluoride (NaF), at 1 mM to the Glc(-) medium also enhanced the inhibitory effects of 10 microM phosphate, whereas 1 mM NaF in the absence of phosphate showed no inhibitory effects on the development of 2-cell embryos to morula and blastocyst stages. From these results, disturbance of glycolysis is a critical reason for the developmental inhibition caused by phosphate in early rat embryos in culture.     

Effects of Phosphonic Acid Analogues of Glycerol-3-Phosphate on the Growth of Escherichia coli

The four-carbon phosphonate, 3,4-dihydroxybutyl-1-phosphonate, is similar to glycerol-3-phosphate in its ability to inhibit cell growth of Escherichia coli strain 8 cultured in low-phosphate synthetic medium supplemented with either succinate or casein hydrolysate as the sole carbon source. The three-carbon phosphonate, 2,3-dihydroxypropyl-1-phosphonate, does not appear to exhibit a similar effect. The inhibition caused by the four-carbon phosphonate differs from that caused by glycerol-3-phosphate in at least three ways. (i) Its inhibitory effect is not offset by the presence of glucose in the culture medium. (ii) It is capable of exerting its inhibitory effect on cells containing an active aerobic glycerol-3-phosphate dehydrogenase. (iii) Its inhibitory effect is maintained in synthetic medium containing high concentrations of inorganic phosphate. The four-carbon phosphonate appears to be bacteriostatic and inhibits the uptake of labeled glycerol-3-phosphate by E. coli strain 8.     

The correlation between glycogen–glycolysis metabolite pyruvate and vancomycin antibiotic productions of Amycolatopsis orientalis grown in glucose medium

The effect of glucose concentration in the growth medium on the relationship between glycolysis, glycogen accumulation and vancomycin production of Amycolatopsis orientalis was investigated depending on the incubation time. After a lag phase, bacterial growth of A. orientalis began and biomass concentration increased continuously up to 36th or 48th hours while glucose concentration in the culture medium was consumed rapidly in the same time of incubation. In addition, increase in glucose concentrations of the growth medium lead to increase intracellular glucose as well as glycerol levels. Intracellular pyruvate levels increased significantly up to 15 g/L while extracellular pyruvate levels with respect to increases in glucose concentration. A positive correlation between glucose kinase activities and glucose concentration was determined during the incubation period. Pyruvate kinase activity increased up to 15 g/L glucose and 48th hour of incubation. As a glycopeptide antibiotic, vancomycin production increased with the increases in glucose concentrations up to 15 g/L. These results indicated that glycogen accumulation with respect to glucose concentration of the growth medium was concomitant with the sporulation of A. orientalis. When the initial glucose concentration exceeded 15 g/L, pyruvate excretions as well as intracellular glycogen and glycerol productions were supported in spite of repression in vancomycin production of A. orientalis.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

Inhibition of protein synthesis by glucose 6-phosphate and fructose 1,6-diphosphate in lysed rabbit reticulocytes and the reversal of inhibition by NAD+.

Glucose 6-phosphate and fructose 1,6-diphosphate inhibit protein synthesis when added to lysed rabbit reticulocytes. Protein synthesis is inhibited 47% with 6 mM fructose 1,6-diphosphate and 86% with 6 mM glucose 6-phosphate. With 0.125 mM NAD⁺, the inhibitory effect of glucose 6-phosphate and fructose 1,6-diphosphate becomes stimulatory. The stimulation of protein synthesis in those assays with NAD⁺ and the phosphorylated sugars is 50% above those assays that contain NAD⁺ alone. The inhibition of protein synthesis by glucose 6-phosphate and the reversal of this inhibition by NAD⁺ occurs at a step before the synthesis of the initial dipeptide, methionyl-valine. These data illustrate the importance of NAD⁺ and the activation of glycolysis in regulating protein synthesis in lysed rabbit reticulocytes.     

Inhibition of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in sea urchin eggs by palmitoyl-coenzyme a and reversal by polyamines

Palmitoyl-CoA inhibited crude glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the eggs of the sea urchin, Hemicentrotus pulcherrimus. Fifty percent inhibition of the glucose 6-phosphate dehydrogenase in the supernatant of unfertilized eggs was obtained with 0.43 ± 0.05 μm palmitoyl-CoA, and of 6-phosphogluconate dehydrogenase with 4.41 ± 0.20 μm palmitoyl-CoA. Also, these enzymes in fertilized eggs 30 min after fertilization were inhibited by palmitoyl-CoA almost as much as in unfertilized eggs. Na-Palmitate, coenzyme A, acetyl-CoA, palmitoylcarnitine, and carnitine failed to exert any inhibitory effect on the activities of these dehydrogenases. The intracellular concentration of long-chain fatty acyl-CoA in unfertilized eggs (3.08 ± 0.33 nmol/10⁶ eggs) was high enough for the inhibition of these enzymes, and decreased following fertilization to a low level (1.49 ± 0.08 nmol/10⁶ eggs 30 min after fertilization). Spermine and spermidine canceled the inhibition of these enzymes by palmitoyl-CoA. In view of the inhibition of glucose 6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase by palmitoyl-CoA, these dehydrogenases in the pentose monophosphate cycle are probably inhibited in unfertilized eggs by long-chain fatty acyl-CoA and released from the inhibited state by both the decrease in the level of long-chain acyl-CoA and the increase in the level of polyamines following fertilization.     

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [¹⁴C]pyruvate to haemoglobin and the rate of efflux of [¹⁴C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent K_m and V_max for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : site-specific inhibition of the photosynthetic carbon reduction cycle

The effects of reduced osmotic potential on the photosynthetic carbon reduction cycle were investigated by monitoring photosynthetic processes of spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts exposed to increased assay medium sorbitol concentrations. CO₂ assimilation was found to be inhibited at 0.67 molar sorbitol by about 60% from control rates at 0.33 molar sorbitol. This level of stress inhibition was greater than that affecting the reductive phase of the cycle; glycerate 3-phosphate reduction was inhibited at 0.67 molar by 27 to 40%. Sorbitol (0.67 molar) inhibited the rate of O₂ evolution at saturating and limiting concentrations of NaHCO₃, and extended the lag phase of O₂ evolution. This indicated that factors which are rate-limiting to the photosynthetic process are adversely affected by reduced osmotic potential.

Analysis of photosynthetic products following CO₂ fixation in 0.33 molar sorbitol and 0.67 molar sorbitol indicated that reduced osmotic potential facilitated increases in the levels of fructose 1,6-bisphosphate and triose phosphates with reductions in glucose 6-phosphate and fructose 6-phosphate, implicating fructose 1,6-bisphosphatase as a site of osmotic stress. Osmotic inhibition of the reductive portion (glycerate 3-phosphate to triose phosphate) of the photosynthetic carbon reduction cycle was partially attributed to feedback inhibition by the product, triose phosphate, on glycerate 3-phosphate reduction. A saturating concentration of ribose 5-phosphate partially overcame osmotic inhibition of CO₂-supported O₂ evolution, indicating another but apparently less severe site of stress inhibition in the sequence of ribose 5-phosphate to glycerate 3-phosphate.

     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Studies on the energy metabolism ofRana ridibunda erythrocytes

Summary Rana ridibunda erythrocytes have a complete sequence of glycolytic enzymes but not the tricarboxylic acid cycle enzymes.The steady state contents of the glycolytic intermediates were measured in quick frozenRana ridibunda erythrocytes. A comparison of the mass action ratios with the equilibrium constants for the glycolytic reactions showed that phosphoglucomutase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, phosphoglycerate mutase and enolase reactions are all near equilibrium whilst hexokinase, phosphofructokinase and pyruvate kinase are displaced from equilibrium.The steady state contents of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate have been measured during various periods up to 4 h of incubation of erythrocytes in the presence of glucose. In the incubation experiment glycolysis had been stimulated by the high pH-value of the medium. After 4 h of incubation 3 patterns of changes can be distinguished. One group of intermediates (glucose, glucose 6-phosphate, 2-phosphoglycerate and inorganic phosphate) in which the concentration of metabolites was lower than the zero time values. A second group of metabolites (fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and AMP) in which the concentration was about the same at zero time and after 4 h of incubation. The metabolites of the third group (dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate, 3-phosphoglycerate, pyruvate, lactate, ADP, ATP and glucose 1-phosphate) all increased their content during the 4 h of incubation in comparison to the zero time values.From the results it appears that in the amphibian erythrocyte glycolysis seems to be similar to that of mammalian erythrocytes as far its control and organisation is concerned down to the level of PEP, with the exception of the low concentration of phosphoglycerate compounds.Abbreviations 2,3DPG 2,3-diphosphoglycerate - EDTA [ethylene dinitrilo]-tetra-acetic acid - P _i inorganic phosphate - DTNB 5,5-dithio-bis-(2-nitrobenzoic acid) - PEP phosphoenolpyruvate - RBC red blood cells     

Implications of the simultaneous occurrence of hepatic glycolysis from glucose and gluconeogenesis from glycerol.

Glycolysis from [6-(3)H]glucose and gluconeogenesis from [U-(14)C]glycerol were examined in isolated hepatocytes from fasted rats. A 5 mm bolus of glycerol inhibited phosphorylation of 40 mm glucose by 50% and glycolysis by more than 60%, and caused cellular ATP depletion and glycerol 3-phosphate accumulation. Gluconeogenesis from 5 mm glycerol was unaffected by the presence of 40 mm glucose. When nonsaturating concentrations of glycerol (< 200 microm) were maintained in the medium by infusion of glycerol, cellular ATP concentrations remained normal. The rate of uptake of infused glycerol was unaffected by 40 mm glucose, but carbohydrate synthesis from glycerol was inhibited 25%, a corresponding amount of glycerol being diverted to glycolytic products, whereas 10 mm glucose had no inhibitory effect on conversion of infused glycerol into carbohydrate. Glycerol infusion depressed glycolysis from 10 mm and 40 mm glucose by 15 and 25%, respectively; however, the overall rates of glycolysis were unchanged because of a concomitant increase in glycolysis from the infused glycerol. These studies show that exposure of hepatocytes to glucose and low quasi-steady-state concentrations of glycerol result in the simultaneous occurrence, at substantial rates, of glycolysis from glucose and gluconeogenesis from the added glycerol. We interpret our results as demonstrating that, in hepatocytes from normal rats, segments of the pathways of glycolysis from glucose and gluconeogenesis from glycerol are compartmentalized and that this segregation prevents substantial cross-over of phosphorylated intermediates from one pathway to the other. The competition between glucose and glycerol implies that glycolysis and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis may indicate channelling within the cytoplasm of individual hepatocytes.     

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP^2?. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP^4? and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.     

Chloroplast phosphofructokinase in the green alga, Dunaliella marina: partial purification and kinetic and regulatory properties

The aim of this work was to study the pathway(s) of sugar phosphate metabolism in chloroplasts of the unicellular green alga, Dunaliella marina (Volvocales). Phosphofructokinase, detectable in crude cell extracts, copurifled with intact chloroplasts on sucrose density gradients. In isolated chloroplasts, phosphofructokinase activity displayed latency to the same degree as chloroplast marker enzymes. From the quantitative distribution of enzyme activities in fractionated cells, it is concluded that there is an exclusive localization of phosphofructokinase in chloroplasts. In addition, no separation into multiple forms could be achieved. For the study of regulatory properties, chloroplast phosphofructokinase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. The pH optimum of the enzyme activity was 7.0 and was not altered with varying concentrations of substrates or low-molecular-weight effectors. Fructose 6-phosphate showed a sigmoidal saturation curve whose shape was further changed with varying protein concentrations of the preparation. The second substrate, ATP, gave a hyperbolic saturation curve with a Michaelis constant of 60 μm. At a Mg²⁺ concentration of 2.5 mm, ATP concentrations exceeding 1 mm inhibited the enzyme in a positive cooperative manner. The same type of inhibition was observed with other phosphorylated intermediates of carbon metabolism, the most efficient being phosphoenolpyruvate, glycolate 2-phosphate, glycerate 3-phosphate, and glycerate 2-phosphate. Inorganic phosphate was the only activator found for phosphofructokinase. With nonsaturating fructose 6-phosphate concentrations, P_i activated in a positive cooperative fashion, while no activation occurred with saturating fructose 6-phosphate concentrations. In the presence of either an activator or an inhibitor, the sigmoidal shape of the fructose 6-phosphate saturation curve was altered. Most notably, the activator P_i could relieve the inhibitory action of ATP, phosphoenolpyruvate, glycerate 3-phosphate, glycerate 2-phosphate, and glycolate 2-phosphate. Based on these experimental findings, the regulatory properties of D. marina chloroplast phosphofructokinase are discussed with respect to its playing a key role in the regulation of chloroplast starch metabolism during a light/dark transition. All available evidence is compatible with the interpretation that phosphofructokinase is active only in the dark thus channeling starch degradation products into glycolysis.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.