Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development.

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.

pH sensitivity of the thrombin-induced rise in fructose 2,6-bisphosphate content of human platelets.

The stimulation of human platelets with thrombin results in a rapid and sustained increase in the fructose 2,6-bisphosphate content which may play an important role in the potentiation of glycolytic flux induced by the agonist. The investigation of the effect of pH on thrombin-induced rise in platelet fructose 2,6-bisphosphate content is reported here. The results indicate that the early intracellular alkalinization which follows platelet stimulation may contribute to mediate the positive effect of thrombin on the regulatory metabolite.

Fructose 2,6-bisphosphate in human erythrocytes.

The fructose 2,6-bisphosphate concentrations in unwashed, washed, and leukocyte-free erythrocytes were compared. The concentration in washed red cells was 31 +/- 15 pmol per ml of cells (mean +/- S.D., n = 6). The concentration in unwashed erythrocytes was at least twofold higher, but the value in washed red cells was not due to leukocyte contamination because it did not decrease further when washed cells were passed through an Imgard column, which would have removed any remaining leukocytes. No platelets were detected among the washed erythrocytes. Thus, the concentration in erythrocytes after washing was ascribed solely to these cells. The fructose 2,6-bisphosphate concentration did not change when the glycolytic activity varied with pH, indicating that this compound is not involved in the regulation of carbohydrate metabolism in erythrocytes under these conditions.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate in rabbit erythroid cells during differentiation.

Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity markedly decrease during differentiation of rabbit erythroid cells, being higher in erythroblasts (654 +/- 97 pmol/10(9) cells; 238 +/- 81 U mu/10(9) cells) than in reticulocytes (40 +/- 15 pmol/10(9) cells; 11 +/- 3 U mu/10(9) cells) and much higher than in mature erythrocytes (10 +/- 0.8 pmol/10(9) cells; 2 +/- 1 U mu/10(9) cells). The enzymatic activities involved in glucose 1,6-bisphosphate metabolism also decrease, but the levels of aldohexose 1,6-bisphosphates remain essentially constant during differentiation of erythroid cells.

Mechanism of thrombin-induced rise in platelet fructose 2,6-bisphosphate content. Studies using phorbol myristate acetate, dioctanoylglycerol and ionophore A23187.

The mechanism by which thrombin increases platelet fructose 2,6-bisphosphate content was investigated. The action of thrombin was mimicked by phorbol 12 myristate 13-acetate and 1,2-dioctanoylglycerol. Ca2+ with A23187 potentiated the action of both these compounds. The action of thrombin required mobilization of intracellular and extracellular Ca2+ and was not decreased by indomethacin. This study suggests that protein kinase C activation and Ca2+ mobilization are both involved in the activation of glycolysis by thrombin.

Fructose 2,6-bisphosphate in rat erythrocytes. Inhibition of fructose 2,6-bisphosphate synthesis and measurement by glycerate 2,3-bisphosphate.

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate in avian and mammalian erythroid cells.

Chicken erythrocytes contain fructose 2,6-P2 at a concentration (0.6 nmol/10(9) cells) lower than that of glucose 1,6-P2 (5.4 nmol/10(9) cells) and similar to that of 2,3-bis phosphoglycerate (1.2 nmol/10(9) cells). In chick embryo erythrocytes the content of both bisphosphorylated hexoses is much lower. They begin to increase at hatching and reach chicken values in a few days. Fructose 2,6-P2 at microM concentration activates phosphofructokinase from chicken erythrocytes and releases the inhibition produced by ATP, 2,3-bisphosphoglycerate and inositol hexaphosphate. Glucose 1,6-P2 has similar effects but at much greater concentration. Rabbit reticulocytes contain fructose 2,6-P2 at a concentration (39 pmol/10(9) cells) much lower than that of glucose 1,6-P2 (74 nmol/10(9) cells).

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate in erythrocytes during chicken development.

In contrast to mammalian erythrocytes, chicken erythrocytes contain fructose 2,6-bisphosphate at levels (0.5 nmol/10(9) cells) similar to those of 2,3-bisphosphoglycerate (1.2 nmol/10(9) cells) and slightly lower than those of glucose 1,6-bisphosphate (5.2 nmol/10(9) cells). In chick embryo erythrocytes the levels of both fructose 2,6-bisphosphate and glucose 1,6-bisphosphate are much lower. They begin to increase at hatching and reach the levels in chicken in a few days.

Fructose 2,6-bisphosphate in human platelets: its possible role in the control of basal and thrombin-stimulated glycolysis.

Human platelets contain fructose 2,6-bisphosphate, 6-phosphofructo-l-kinase (ATP: D-Fructose-6-phosphate-1-phosphotransferase, E.C.2.7. 1.11), the rate-limiting enzyme in platelet glycolysis appear to be significantly activated by physiological concentration of the compound, suggesting for fructose 2,6-bisphosphate a key regulatory role in the control of the glycolytic flux. Incubation of human platelets with thrombin results in a parallel and rapid increase of fructose-2,6-bisphosphate levels and glycolytic flux, suggesting that the compound may also be involved in the enhancement of glycolysis elicited by the stimulating agent.

Increase of intraerythrocytic fructose-1,6-diphosphate after incubation of whole human blood with fructose-1,6-diphosphate.

The incubation of whole blood with fructose-1,6-diphosphate (FDP) entails a statistically significant increase of intraerythrocytic FDP together with a decrease of blood glucose. The increase is not significant when equimolar amounts of fructose plus twice molar phosphate are used. The effect of FDP is decreased in the presence of an excess of oxygen. FDP added to the whole blood is removed from plasma by the activity of plasma enzymes and by the presence of blood cells as well. No specific interaction of FDP with plasma proteins seems to occur and the effects of FDP addition last longer than is compatible with the presence of FDP in the plasma.

Phosphate compounds in the erythrocytes of newborns with ABO systems haemolytic disease.

The following phosphate compounds of the erythrocyte acid-soluble fraction were subjected to chromatographic separation: ADP, ATP, adenylo-diphosphoglycerate, 2,3-diphosphoglycerate, hexose monophosphate, hexose diphosphate. In each of the fractions total phosphorus, and in fraction II inorganic phosphorus, were estimated. The material was derived from ten newborns with haemolytic disease as a result of ABO incompatability and from ten full-term healthy newborns, just after birth. The concentration of the compounds assayed, except for 2,3-DPG (the values in both groups were similar) was higher in the erythrocytes from affected newborns, but lower than that found in the material derived from the newborns with Rh incompatibility. It is suggested that the metabolism of erythrocytes of the newborns with haemolytic ABO disease may be somewhat different from that in Rh incompatibility cases because in the former the haemolysis is weaker, the anaemia is less pronounced and the tissue hypoxia is of a smaller degree.

Enzymatic pattern of glucose metabolic pathways in pyruvate kinase-deficient erythrocytes.

It has been shown that in some cases of congenital non-spherocytic haemolytic anaemia (CNSHA) with pyruvate kinase deficiency, the primary defect may be related to diminished magnesium-stimulated ATPase activity, followed by elevation of the erythrocyte ATP level. ATP as the end product of glycolysis inhibits by negative feedback control the activities of key glycolytic enzymes involved in energy production, i.e. pyruvate kinase (PK) and phosphofructokinase (PFK). Erythrocyte-deficient PK, however, is insensitive to the stimulating effect of fructose 1,6-diphosphate (FDP), which is normally a positive effector of PK. Both competing effectors, i.e. ATP and FDP, seem to show specific interaction. PK, inactive in the presence of high concentrations of ATP, seems to lose its sensitivity to FDP. This effect persists until ATP molecules are present in excess. In vitro incubation of deficient PK with ATPase resulted in increased PK activity as well as in recovery of its sensitivity to the stimulating effect of FDP. The same effects were obtained in vivo by administering magnesium levulinate intravenously to CNSHA patients with PK deficiency. This may indicate that magnesium ions stimulate deficient ATPase activity and lead to diminution of ATP as a negative effector for other regulatory enzymes.