Hg. Hers

Inactivation of Phosphofructokinase-2 By Cyclic Amp-dependent Protein-kinase

Abstract Treatment with the catalytic subunit of cyclic AMP-dependent protein kinase induced the following modifications in the kinetic properties of purified phosphofructokinase 2. The affinity for Fru-6-P, the Vmax and the stimulatory effect of Pi were decreased; the inhibitory actions of P-enol-pyruvate and citrate were increased; the pH activity curve, measured in the presence of 5 mM Fru-6-P and 5 mM Pi was modified in the respect that the peak of activity normally measured at pH 6.6 was abolished whereas no effect of the treatment was observed at pH 8. Similar changes in the properties of phosphofructokinase 2 were also observed in a crude preparation obtained from hepatocytes incubated with glucagon.

The control of glycogen synthesis in the liver

Abstract In the liver, glycogen synthetase exists in two forms, one of them (a) active, the other (b) inactive in the ionic conditions which exist in the cell. These two forms are interconvertible, presumably by phosphorylation and desphosphorylation under the action of a specific kinase and phospharylation and respectively, It seems probable that these two enzymes operate simultaneously and that the level of the synthetase a in the liver results from the addition of their antagonistic effects. In normal mice, glycogen synthetase is predominantly in the b form. It is converted into a within 3 to 5 min after the intravenous administration of glucose or within 2 or 3 hr after the administration of glucocorticoids. The a enzyme can then be reconverted into b within 1 to 3 min after the administration of glucagon, epinephrine or cyclic AMP. The effect of these various effectors has also been demonstrated in vitro, mostly thanks to the use of liver extracts from which glucose had been removed by gel filtration through a Sephadex column. The complete activation of glycogen synthetase in vitro requires the presence of salts. In their absence, synthetase b is converted into a form which has the kinetic properties of synthetase a but which is less active. Glucose markedly enhances the activation, both in the presence and in the absence of salts while glycogen is an inhibitor in the presence of salts only. The affinity of the synthetase phosphatase for glucose is decreased when glycogen is present. A stimulation of the phosphorylase phosphatase by glucose has also been observed. The treatment of mice by glucocorticoids induces the appearance in the liver of a synthetase phosphatase that is less sensitive to glucose stimulation and to glycogen inhibition than normally. No effect of the treatment on the system that inactivates glycogen synthetase could be demonstrated. An effect of cyclic AMP on the synthetase kinase is easily demonstrable in a liver extract in which the synthetase has been previously activated either by glucose or by glucocorticoids; a half-maximal effect has been obtained with a concentration equal to 2 × 10−7 m cyclic AMP. The two main effectors that act antagonistically on the glycogen synthetase and glycogen phosphorylase appear to be glucose and cyclic AMP; thanks to their action, the synthesis of glycogen is inhibited while its degeneration is stimulated and vice versa.

Autosomal Form of Phosphorylase-kinase Deficiency in Man - Reduced Activity of the Muscle Enzyme

Abstract A muscle biopsy from a boy with the autosomal form of phosphorylase kinase deficiency has been analysed. The glycogen content was higher than normal; phosphorylase was mostly in the b form, and the activity of phosphorylase kinase was undetectable at pH 6.8 and reached about 15 % of the mean control value at pH 8.3. The residual activity could be enhanced by trypsin and inhibited by EGTA. Cyclic AMP-dependent and independent protein kinases were normally active.

Glycogen-Storage Diseases: Type II and Type VI Glycogenosis

Publisher Summary This chapter discusses clinical condition known as glycogen-storage disease, which may probably result from a variety of causes but mostly from the genetic defect of a definite enzyme. An inborn lysosomal disease is the situation resulting from the congenital defect of one of the hydrolases normally present in lysosomes. The reason for the very high concentration of glycogen in the muscle of patients with Pompes disease is not understood. In the absence of a well-defined enzymatic defect, the only chemical data which differentiates Type VI from normal is the level of glycogen in the liver. The mean value of liver phosphorylase activity in the cases with Type VI is somewhat lower than in the other groups, although not significantly different from normal. It may be that the numerous cases of Type VI glycogenosis with a low activity of liver phosphorylase constitute a separate disease, which, at present, cannot be clearly delineated because of the large overlapping of phosphorylase activities in the different groups.

The Mucopolysaccharidoses as Lysosomal Diseases

In this paper, we briefly summarize the evidence that has accumulated during the recent years indicating that the diseases known as mucopolysaccharidoses are due to the primary defect of one acid hydrolase normally present in lysosomes.

An Unusual Case of GM2-Gangliosidosis with Deficiency of Hexosaminidase A and B

In this report, we briefly describe a patient with GM2-gangliosidosis having a deficiency in both isoenzymes of N-acetyl-β-hexosaminidase and displaying several clinical, ultrastructural and enzymatic particularities.

Identification of a Purine-specific 5'-nucleotidase in Human-erythrocytes

In human erythrocytes, a pyrimidine 5′-nucleotidase has been characterized (Paglia & Valentine, Curr. Top. Hematol 3:75, 1980) but the enzyme(s) responsible for the dephosphorylation of AMP and IMP had hitherto not been identified. Haemolysates in 10 mM Tris-maleate buffer pH 7, incubated with 1 mM [14C] IMP, produced [14C] inosine at the rate of approx. 2 μmol/h per g of Hb. The enzymic activity was purified around 3000-fold in 3 steps : (1) batchwise binding to DEAE cellulose, followed by washing with 10 mM Tris-maleate pH 6.5 to remove Hb; (2) column separation from acid phosphatase and pyrimidine 5′-nucleotidase by elution with a NaCl gradient in the same buffer, which released the purine 5′-nucleotidase at 0.2 M NaCl; (3) adsorption on blue-sepharose, followed by elution with 2 M NaCl. The enzyme was Mg++-dependent and displayed a broad pH optimum around pH 7. It was maximally active with IMP and hydrolysed GMP at 75 % and AMP at 10 % of the rate of IMP. Km for the three nucleotides was around 0.1 mM. At 0.25 mM AMP, the enzyme activity was 80 % inhibited by 5 mM Pi and stimulated 8-fold by 5 mM 2,3-bis-P-glycerate, half maximal stimulation being obtained at 0.5 mM of this compound. In contrast with rat liver cytosolic 5′-nucleotidase, the erythrocyte enzyme was not stimulated by ATP or GTP. Supported by FRSM.

The Control of Liver Phosphofructokinase by Fructose 2,6-Bisphosphate

One major role of the liver is to form glucose from lactate and other precursors through gluconeogenesis, a process which operates both in the fed and the fasted animal. The reverse conversion is catalyzed by glycolysis and the simultaneous operation of these two metabolic pathways would be responsible for a futile recycling of metabolites.

Pathways of Adenine-nucleotide Catabolism in Human-erythrocytes

Adenine nucleotide catabolism in erythrocytes incubated for 4h under physiological conditions is very low as shown by a production of hypoxanthine (HX) of 4.2 ± 0.7 nmol/h per g of packed cells (mean ± SEM for n=8). It is not influenced by maximal inhibition of adenosine (Ado) deaminase by 10 μM deoxycoformycin (dCF) and of Ado-kinase by 10 μM iodotubercidin (ITu) indicating that it proceeds by deamination of AMP, followed by dephosphorylation of IMP, and that there is no recycling between AMP and Ado. Suppression of glucose increases AMP 30-fold and the production of HX 20-fold. dCF decreases this production by 75 % and provokes an accumulation of Ado, demonstrating a dephosphorylation of AMP. ITu enhances this accumulation, indicating a recycling of Ado under glucose deprivation which reaches approx. 20 nmol/h per ml of cells. Alcalinisation of the incubation medium in the presence of glucose increases AMP 5-fold and the production of HX 15-fold. dCF has no effect on this production, demonstrating that it results from a deamination of AMP. ITu also evidences recycling of Ado under this condition, reaching 25 nmol/h per ml of cells. The 2-fold elevation of intracellular Pi during glucose deprivation and its 50 % decrease during alcalinisation, as well as experiments in which extracellular Pi was modified, indicate that the dephosphorylation of AMP is mainly responsive to variations of AMP, whereas its deamination is more sensitive to modifications of Pi.Supported by FRSM.