Phosphofructokinase during haemopoiesis of the rainbow trout ("Salmo gairdneri R."): its isoenzymatic forms.

Phosphofructokinase (EC 2.7.1.11) from trout haemopoietic cells and erythrocytes exhibits biphasic behaviour, with respect to fructose 6-phosphate and MgATP in extracts filtered through Sephadex G-25. Two different values of Hill coefficient and S0.5 have been found for each cellular population. Two forms of the enzyme with high and low affinity for the substrates, were obtained after affinity chromatography in each cellular population. The kinetic behaviour of these forms is different and may be due to a distinct composition and/or proportion and contribution of phosphofructokinase isoenzymes in both types of cells.

Role of phosphofructokinase during trout haemopoiesis: physiological regulation of glycolysis.

1. The regulatory properties of phosphofructokinase (PFK) has been investigated in two cellular population representatives of trout haemopoiesis; haemopoietic cells (capable of replication and differentiation) and erythrocytes (highly specialized cells). 2. The intracellular levels of substrates and effectors have been quantified and their effect on PFK activity determined. 3. Fructose 1,6-bisphosphate anc cyclic AMP show a higher activation of the PFK from haemopoietic cells than the enzyme from erythrocytes. 4. AMP and phosphoenolpyruvate act as activators of the haemopoietic cell PFK while for erythrocytes PFK, AMP is an inhibitor and phosphoenolpyruvate does not display any effect. 5. Citrate inhibits PFK activity from haemopoietic cells but was not assayed in erythrocytes since it was not detected in these cells. 6. The differences in PFK regulation in both cellular populations may be attributed to the intracellular levels of the effectors and/or different isoenzymatic patterns. 7. The different regulation of PFK together with the higher enzymatic activity of PFK and pyruvate kinase from haemopoietic cells are related to the higher glycolytic flux that exhibits the haemopoietic cells. 8. The results shown in this investigation allow us to conclude that PFK has a specific role depending on the energetic requirements of the cellular population in which the enzyme is present. 9. The requirements are related to the physiological function of each type of cell.

Thermic and pH modulation of phosphofructokinase during trout (Salmo gairdneri R.) haemopoiesis: influence of fructose 6-phosphate.

Thermic and pH modulation of phosphofructokinase (EC 2.7.1.11) activity with respect to fructose 6-phosphate has been studied comparatively in trout (Salmo gairdneri R.) haemopoietic cells and erythrocytes. Phosphofructokinase of both cellular populations displays a biphasic kinetic behaviour with respect to fructose 6-phosphate at two values of pH and temperature. In haemopoietic cells, when pH decreases the enzyme-substrate affinity increase while an opposite effect is found in erythrocytes. Decreases in temperature act as a positive modulator in haemopoietic cells while in erythrocytes this effect is observed only at low fructose 6-phosphate concentrations. Therefore a different pH and temperature modulation of phosphofructokinase during trout haemopoiesis has been established.

Metabolic regulation of glycolysis in sea bass (Dicentrarchus labrax L.) muscle. I. Kinetic study and characteristic modulators of pyruvate kinase.

White muscle pyruvate kinase from sea bass presents positive cooperativity with respect to PEP substrate. The enzyme is regulated by F-1.6-P2 and L-Phenylalanine. The activator effect of F-1.6-P2 in experiments carried out for the substrate PEP with crude extract seems to indicate that the enzyme is activated in vivo by this compound. The enzyme was not inhibited by either alanine or ATP but was inhibited by L-phenylalanine. Therefore this enzyme presents kinetic and regulatory properties similar to those of the mammalian isozyme M2.

Erythroid 5-aminolevulinate synthetase from trout (Salmo gairdneri R.).

A mitochondrial and cytosolic erythroid ALA-synthetase have been found in trout. The polypeptide existent in the cytosol is probably a precursor of the 90,000 mol. wt mitochondrial ALA-synthetase. The erythroid ALA-synthetase is about 20,000 mol. wt larger than the hepatic enzyme. The differences in mol. wt and catalytic properties between erythroid and hepatic enzyme support the existence of two forms of the ALA-synthetase in teleostei.

Influence of ATP and magnesium on phosphofructokinase from sea bass (Dicentrarchus labrax L.) liver.

Glycolytic enzyme phosphofructokinase (PFK) from sea-bass liver shows inhibition for ATP4- and MG-ATP2-, and ATP4- is a competitive inhibitor with respect to MG-ATP2-. Free Mg2+ behaves as a mixed inhibitor on the kinetic with respect to the true enzyme substrate Mg-ATP2-, and eliminates the inhibition effect of this substrate. The kinetics with respect to Mg-ATP2- at non-inhibiting concentrations is not visibly affected by temperature of pH variation. The inhibiting effect of Mg-ATP2- is more marked at 22 and 10 degrees C (of three assayed temperatures 22, 15 and 10 degrees C and at physiological pH 6.8) as opposed to the maximum activity pH (8.0).

Some comparative properties of pyruvate kinase in haematopoietic cells and erythrocytes from rainbow trout (Salmo gairdneri R).

1. Temperature acts as a pyruvate kinase regulator in haematopoietic cells and erythrocytes. 2. Fructose-1,6-biphosphate and alanine act as allosteric modulators of pyruvate kinase in haematopoietic cells, while in erythrocytes although fructose-1,6-biphosphate exerts also allosteric effect, alanine appears to be a competitive inhibitor. ATP (1.0 mM) does not exert any clear effect on pyruvate kinase of both cellular populations. 3. The level of specific activity of pyruvate kinase in haematopoietic cells is 40-fold that of PK from erythrocytes.