Pyruvate kinase and the "high ATP syndrome"

The erythrocytes of a patient with the so-called "high ATP syndrome" were characterized by a high ATP content and low 2,3-diphosphoglycerate level. The pyruvate kinase activity was specifically increased (about twice the normal level). After separation of the erythrocytes according to age by discontinuous Percoll density centrifugation, the pyruvate kinase activity was found to be increased in all Percoll fractions. Pyruvate kinase of the patient's cells was characterized by a decreased K0.5 for the substrate phosphoenolpyruvate and no inhibition by ATP. The Michaelis constant (Km) value for ADP, the nucleotide specificity, the thermostability, pH optimum, and immunological specific activity were normal. It is concluded that the high pyruvate kinase activity is due to a shift in the R(elaxed) in equilibrium T(ight) equilibrium to the R(elaxed) form.     

A combined system for the study of glutathione metabolism in erythrocytes.

A combined system for measuring glutathione stability, hexose monophosphate shunt activity, glycine incorporation into glutathione, glucose consumption and lactate formation in erythrocytes has been devised. In severe glucose-6-phosphate dehydrogenase deficiency associated with chronic hemolytic anemia, extremely small amounts of glucose were metabolized via the hexose monophosphate shunt, glutathione stability decreased, and glycine incorporation into glutathione increased. However, in mild glucose-6-phosphate dehydrogenase deficiency with no clinical manifestation, glutathione stability and recycling via the hexose monophosphate shunt decreased and hexose monophosphate shunt activity decreased slightly. In glucosephosphate isomerase deficiency, more than half of glucose was metabolized via the hexose monophosphate shunt, while Embden-Meyerhof pathway activity and recycling via the hexose monophosphate shunt decreased. In a splenectomized patient with pyruvate kinase deficiency with reticulocytosis, high hexose monophosphate shunt activity, glucose consumption and lactate formation were observed; but in pyruvate kinase deficiency without splenectomy, decreased glutathione stability, low glucose consumption and lactate formation were observed.These results indicate that one abnormal enzyme may exert its influence on several enzyme systems and that it is valuable to investigate the metabolism of erythrocytes using this system in different enzymopathies as well as different variants of one enzyme deficiency.     

Hereditary erythrocyte pyruvate kinase deficiency: molecular and functional studies of four mutant PK variants detected in Spain

The mechanisms of the defect in enzyme activity and the functional anomalies have been studied for four different mutant erythrocyte pyruvate kinase (PK) variants found in Spain.In two cases the PK variants were heat stable, and their antigenic concentration was nearly normal in the patients' red cells: the defect in activity was due to decreased catalytic activity of the mutant molecules. In the two other observations the mutant PK variants were heat-labile, and their antigenic concentration was reduced in the red cells: the defect in activity was entirely due to this decrease of the enzyme molecule concentration in one case, and to decrease of the enzyme molecule concentration plus decrease of catalytic activity of these mutant molecules in the other case.Kinetic properties were normal for one variant and drastically modified in the three other variants. These latter variants showed decreased apparent affinity for PEP, which cannot be simply explained by a shift of the R α T allosteric equilibrium towards the T form: the results of the study of ATP inhibition and FDP activation, indeed, did not correspond to the expected results for a PK enzyme present in a predominant T form.We point out the heterogeneity and the complexity of the anomalies of the mutant PK variant, most likely reflecting the diversity of the possible punctual mutations of the PK structural gene.     

Significance of the electrophoretic modifications of defective pyruvate kinase variants. Study of six new observations.

Six new defective pyruvate kinase variants have been characterized in patients suffering from chronic hemolysis. Partially purified enzyme variants exhibited various anomalies from immunological, kinetic, stability and electrophoretic points of view. The significance of the electrophoretic anomalies has been interpreted in view of the normal post-synthetic maturation of the precursor enzyme L'4 into L2L'2 and L4, and the ability of trypsin to induce in vitro the transition L'4 leads to L4 has been tested. One defective enzyme existed in a single L'4 form and could not be transformed by trypsin into L4. In three cases slow-moving L'4 and L2L'2 forms were transformed by trypsin into an abnormal slow-moving L4 form. In the last two observations the L'4 and L2L'2 forms exhibited normal mobility and were normally transformed by trypsin into L4. The relevance of these data to the functional anomalies of the defective variants and to the nature of the primary genetic anomaly giving rise to the congenital defects in erythrocyte pyruvate kinase is discussed.     

Mechanisms of the acquired erythrocyte enzyme deficiencies in blood diseases.

Acquired enzymatic activity defects of erythrocyte pyruvate kinase, glucose phosphate isomerase and phosphofructokinase have been studied in patients with acute myeloid leukemias, sideroblastic refractory anemias and unclassified acquired dyserythropoiesis. 6 patients with acute myeloid leukemia had a lowered erythrocyte pyruvate kinase activity; in 5 of them the concentration of the "pyruvate kinase"-antigen was parallely decreased, in such a manner that the ratio enzyme activity/immunologic reactivity (i.e. the molecular specific activity) was normal. In 1 patient with acute leukemia, 4 with refractory anemia and 1 with acquired dyserythropoiesis the defect of the pyruvate kinase activity was associated with a normal antigen concentration (and, therefore, the molecular specific activity in whole hemolysate was lowered). The enzyme activity was restored by incubation with SH reagents in two cases and by partial purification as often as it was performed. The electrofocusing pattern of erythrocyte pyruvate kinase was normal in both these types of defects. In two patients with so-called "acquired dyserythropoiesis" an erythrocyte glucose phosphate isomerase deficiency has been detected; in both the cases it was associated with a parallel decrease of the antigen concentration. The residual enzyme had a normal electrofocusing and electrophoretic pattern and a normal heat stability; the enzyme activity could not be restored by any treatment. In 1 patient with erythroleukemia and in 1 other with acquired dyserythropoiesis the erythrocyte phosphofructokinase activity was lowered. The enzyme activity was not restored by cross incubation in isologous plasma or by the SH reagents. In one case immunologic study could be performed, indicating that the enzyme defect was mainly due to the decreased ratio of the muscle type subunit of the erythrocyte phosphofructokinase. The electrofocusing pattern of deficient phosphofructokinases was normal. Finally, we point out the probable existence of several direct mechanisms, genetic and post translational, accounting for the acquired enzyme defects of red blood cells in various blood disorders.     

Heterogeneity of erythrocyte pyruvate kinase deficiency and related metabolic disorders in patients with hematological diseases.

In several patients suffering from congenital non-spherocytic hemolytic anemia or from malignant hemotological disorder associated with erythrocyte pyruvate kinase (PK) deficiency, a metabolic study has been carried out involving the following biochemical determinations: assay of red cell enzyme activities; estimation of glucose consumption; measurement of the rate of glycolytic intermediates; and, in some cases, enzyme purification and characterization of the PK variant. Metabolic equilibrium most probably does not depend on kinetic characteristics of PK molecules. Furthermore, the data obtained allow separation of cases with congenital non-spherocytic hemolytic anemia (hereditary defect) and acquired PK deficiencies.     

Gas chromatographic and mass spectrometric studies on urinary organic acids in a patient with congenital lactic acidosis due to pyruvate decarboxylase deficiency.

Detailed studies, using gas chromatography and mass spectrometric methods, of the urinary organic acids excreted by a patient with proven pyruvate decarboxylase deficiency are reported. In addition to the greatly-increased levels of lactate and pyruvate, marked elevation in the levels of 2-oxoglutaric, malic, and isocitric acids were observed, with associated increases 2-hydroxyglutaric, fumaric, succinic, and glyceric acids, and reduced citric acid excretion. The levels of excretion during clinically static and acute periods are compared to those in a normal neonate and normal infants. The metabolites observed indicate a probable defect in the oxidation of pyruvate by pyruvate dehydrogenase and suggest the presence of secondary defects in the tricarboxylic acid cycle. Studies of this type may enable the relatively rapid identification of the probable underlying enzyme deficiency in cases of congenital lactic acidosis, prior to confirmatory enzyme studies.     

In vivo lability of glucose-6-phosphate dehydrogenase in GdA- and Gdmediterranean deficiency

A decreased level of glucose-6-phosphate dehydrogenase might result from decreased rate of synthesis, synthesis of an enzyme of lower catalytic efficiency, increased lability, or a combined mechanism. To test the hypothesis of increased lability, the rate of decline of the enzyme in vivo was measured in three groups of individuals, controls, Gd(-),A-males, and Gd(-), Mediterranean males, by the slope of decline of activity in fractions containing erythrocytes of progressively increasing mean age. These fractions were obtained by ultracentrifugation on a discontinuous density gradient of erythrocyte suspensions free of contaminating platelets and leukocytes.The rate of in vivo decline of pyruvate kinase (another age-dependent enzyme) was also measured and found very similar in the three groups.The in vivo decline of glucose-6-phosphate dehydrogenase was found to follow an exponential rate, with a half-life of 62 days for controls and 13 days for Gd(-),A- erythrocytes. The activity in normal reticulocytes was estimated at 9.7 U and in Gd(-),A- reticulocytes at 8.8 U. These estimates were confirmed by direct measurements in reticulocytes isolated from patients with extreme reticulocytosis.In Gd(-),Mediterranean erythrocytes activity could be demonstrated only in reticulocytes, which were estimated to average 1.4 U. The rate of decline is so extreme that no activity could be detected in mature erythrocytes.These data suggest that the glucose-6-phosphate dehydrogenase deficiency of both the Gd(A-) and the GdMediterranean variant results from different degrees of in vivo instability of the abnormal enzyme.     

Enzymatic and Metabolic Studies on Carbohydrate and Amino Acid Metabolism in Rat Liver during Acute Uraemia

Abstract. Enzyme activities of the glycolytic, gluconeogenic, and hexose monophosphate pathways were measured in the liver of starved rats 12 and 48 hours after bilateral nephrectomy. Control experiments (sham operated rats) revealed that alterations of enzyme activities were not due to uraemia but to starvation. Alanine-aminotransferase and aspartate- aminotransferase activities, however, were significantly elevated in rat liver 48 hours after nephrectomy when compared with sham operated controls. Concentrations of some of the gluconeogenic intermediates (3-phosphoglyceric acid, pyruvate, phosphoenolpyruvate and glucose-6-phosphate) were significantly higher in the liver of uraemic animals. Amino acid analysis showed an increase in only L-alanine concentration. It is suggested that the elevated content of pyruvate in the liver during acute uraemia is due to an inhibition of pyruvate degradation. Together with the elevated pyruvate concentration the increase in L-alanine could be explained as a consequence of the equilibrium of the alanine-aminotransferase reaction; K_app. of the reaction is not changed by uraemia. Increased activities of the transaminases and the elevated concentrations of the other metabolites measured might indicate that in the liver of nephrectomized rats there is enhanced gluconeogenesis from substrates other than pyruvate.     

Glycolytic enzymes of stored granulocytes

A marked reduction of granulocyte chemotactic function accompanies the storage of granulocyte concentrates. Since chemotaxis is energy dependent, we studied energy metabolism in stored neutrophils. We and others have reported that stored neutrophils have a defect in their energy metabolism. We found that defective adenosine triphosphate maintenance in stored neutrophils was occult in resting cells, but was unmasked by an energy-intensive stimulus, phagocytosis. In studies reported here, we sought to determine if defective adenosine triphosphate maintenance during granulocyte storage was related to altered glycolytic enzyme activity. We studied the activity of glycolytic enzymes in fresh and stored, resting and stimulated (opsonized zymosan) neutrophils. The following enzyme activities showed no major changes during storage, in resting or stimulated neutrophils: hexokinase, phosphofructokinase, aldolase, glucose phosphate isomerase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione peroxidase. In contrast, pyruvate kinase activity consistently increased during storage. In 6 units, pyruvate kinase activity increased by 75 percent after 24 hours of storage at room temperature and by 198 percent after 48 hours. The storage-associated increase in pyruvate kinase activity was not inhibited by cycloheximide. Stimulation of neutrophils by phagocytosis of opsonized zymosan also produced striking increases in the pyruvate kinase activity of both fresh and stored cells. Additional studies indicated that the increases in pyruvate kinase activity observed during storage and after phagocytosis were associated with an increase in the availability of pyruvate kinase activity in the supernatant fraction of neutrophil sonicates. Total pyruvate kinase activity in sonicates of neutrophils was unchanged by storage or particle ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normalisation of red blood cell pyruvate kinase in pyruvate kinase deficiency by riboflavin treatment

A patient with an erythrocyte glutathione reductase activity of 50% of the normal value and an abnormal pyruvate kinase (PK) was given 36 mg riboflavin daily for 6 months. The glutathione reductase activity was restored and the abnormal pyruvate kinase was converted to normal. The clinical state of the patient improved. It can be concluded that the abnormality of PK, at least with this patient, is a secondary effect. Therefore, it is suggested that other abnormalities be searched for when an altered PK is detected. Treatment of this abnormality will help the patients more efficiently.     

Cyclic AMP suppresses the inhibition of glycolysis by alternative oxidizable substrates in the heart.

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.     

Pyruvate kinase electrophoresis in normal and pyruvate kinase deficient hemolysates

Two bands of pyruvate kinase activity were demonstrated in normal hemolysates with high voltage electrophoresis at pH 5.3 using cellulose acetate strips.Heterozygotes with red cell pyruvate kinase deficiency also exhibited two bands. Six of ten homozygous patients showed two bands, the remaining four only one band. No mutant enzyme with abnormal electrophoretic mobility could be demonstrated among the ten homozygous individuals with pyruvate kinase deficiency. High voltage electrophoresis may be an additional procedure for the characterization of PK variants in patients with congenital hemolytic anemia associated with PK deficiency.     

Detection of glucose-6-phosphate dehydrogenase deficiency in erythrocytes: a spectrophotometric assay and a fluorescent spot test compared with a cytochemical method

The results of a quantitative spectrophotometric enzyme assay, a fluorescent spot test and a cytochemical assay for glucose-6-phosphate dehydrogenase deficiency were compared systematically. The high sensitivity of the spectrophotometric assay and the fluorescent spot test in the detection of severely deficient individuals was confirmed. For the detection of heterozygote females, however both tests were unreliable; the sensitivities of the fluorescent spot test and the spectrophotometric assay being 32% and 11% respectively. Specificities for both tests were high (99%). Introduction of the ratio of glucose-6-phosphate dehydrogenase and pyruvate kinase (G-6-PD/PK ratio) activities increased the sensitivity of the spectrophotometric assay to nearly 100%. It is concluded that the fluorescent spot test should be used for the diagnosis of G-6-PD deficiency in developing countries; whereas if spectrophotometric enzyme assays are available, the G-6-PD/PK ratio should always be performed. In cases where the ratio is less than 0.70, cytochemical analysis is indicated.     

The measurement of propionyl-CoA carboxylase and pyruvate carboxylase activity in hair roots: its use in the diagnosis of inherited biotin-dependent enzyme deficiencies

Two mitochondrial biotin-dependent enzymes, propionyl-CoA carboxylase and pyruvate carboxylase, are measurable in hair roots. A third biotin-dependent enzyme, beta-methylcrotonyl-CoA carboxylase, was barely detectable in hair roots. The diagnosis of isolated propionyl-CoA carboxylase deficiency was confirmed in hair roots of a known affected patient. This method should be a rapid and accurate method for the diagnoses of the various carboxylase deficiencies, particularly isolated pyruvate carboxylase deficiency in individuals with lactic acidosis, as well as for the assessment of biotin responsiveness in these patients.     

Secretion of pyruvate. An antioxidant defense of mammalian cells

Cells in culture are exposed to marked oxidative stress, H2O2 being one of the predominant agents. Pyruvate and other alpha-ketoacids reacted rapidly, stoichiometrically, and nonenzymatically with H2O2, and they protected cells from its cytolytic effects. All five human and murine cell types studied, both malignant and nonmalignant, released pyruvate at an initial rate of 35-60 microM/h/2.5 X 10(6) cells when placed in 1 ml pyruvate-free medium. After 6-12 h a plateau of 60-150 microM pyruvate was attained, corresponding to concentrations reported for normal human serum and plasma. The rate of pyruvate accumulation was almost doubled in the presence of exogenous catalase, suggesting that released pyruvate functions as an antioxidant. The rate of pyruvate accumulation was dependent on cell number. Succinate, fumarate, citrate, oxaloacetate, alpha-ketoglutarate, and malate were not secreted in significant amounts from P815 cells; export was specific for pyruvate and lactate among the metabolites tested. Extracellular pyruvate was in equilibrium with intracellular stores. Thus, cells conditioned the extracellular medium with pyruvate at the expense of intracellular pyruvate, until homeostatic levels were attained in both compartments. We propose that cells plated at low density in the absence of exogenous pyruvate fail to thrive for two reasons: prolonged depletion of intracellular pyruvate and prolonged vulnerability to oxidant stress.     

Isozyme distribution of hexokinase, phosphofructokinase and pyruvate kinase in lymphocytes from patients with chronic lymphocytic leukemia

The enzyme activities and isozyme distribution of the three glycolytic regulator enzymes hexokinase, phosphofructokinase and pyruvate kinase were studied in lymphocytes of patients with chronic lymphocytic leukemia. Isozyme distribution patterns were determined by kinetic measurements, electrophoresis and immunoprecipitation. The CLL lymphocytes were different from normal non-T lymphocytes with respect to hexokinase residual activity in the presence of glucose-1,6-P2, pyruvate kinase residual activity in the presence of alanine, and phosphofructokinase activity after stimulation by glucose-1,6-P2. No differences could be discerned in enzyme activities between the CLL and the normal T and non-T lymphocytes.     

Characterization of alpha-L-fucosidase from two different families with fucosidosis.

1. Two different families with a different type of fucosidase deficiency are described. 2. In the first family the activity of alpha-L-fucosidase in leucocytes of two patients with fucosidosis type I was about 4 to 8% of the normal value. The activity of alpha-L-fucosidase in the leucocytes of the father and the mother are in the heterozygote range, while a sister of the propositus showed normal values. 3. The activity of alpha-L-fucosidase of the propositus from urine, serum and liver were also severely decreased. The activity of alpha-L-fucosidase in the urine of the parents and the healthy sister of thr propositus were about 5% of the mean normal value. However in the serum these values were above 50%. 4. The KM value for alpha-L-fucosidase from leucocytes of the patient was increased about 10 times and in serum this value was even higher. The KM values from the enzyme of the parents were in the normal range. 5. The abnormal enzyme from the propositus is unique in its thermal behaviour since after heating its activity increased. 6. In the second fanily the activity of alpha-L-fucosidase in the leucocytes of the patient is about 30% of the mean normal value, while the arylsulphatase A activity is also decreased (25% of the mean normal value). 7. The activity of alpha-L-fucosidase from the leucocytes of the father and the healthy brother are about 50% of the mean normal level, while the enzyme of the mother showed a normal activity. 8. The alpha-L-fucosidase activity in the urine and the liver of the propositus is also decreased. The serum enzyme activity however was in the normal range. 9. The KM value of alpha-L-fucosidase and heat stability of the enzyme of the patient were normal. In the leectrophoretic pattern of the whole family one bond was missing.     

Red Cell Glycolysis in a Case of 3-Phosphoglycerate Kinase Deficiency

Abstract. A severe deficiency in red cell 3-phosphoglycerate kinase was observed in a 62-year-old woman with haemolytic anaemia. Compared with a normal “young” red cell population with the same degree of reticulocytosis (6–7%) the 3-phosphoglycerate kinase activity was reduced to 27%. A concomitant decrease of hexokinase and pyruvate kinase (both reduced by about 30%) was observed. The activities of glucoses-phosphate dehydrogenase, phosphofructokinase, fructose-1,6-di-phosphate aldolase, gIyceraIdehyde-3-phosphate dehydrogenase and lactate dehydrogenase were slightly increased (7 to 15%). The total glycolytic output of the deficient cells was decreased by 28% at pH 7.0, by 36% at pH 7.4 and by 34% at pH 7.6. Compared with a normal “adult” red cell population the 3-phosphoglycerate kinase activity was reduced to 42% of the control values. Hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase activities were increased by approximately 20–50%. Phosphofructokinase activity was unchanged and pyruvate kinase only slightly increased. The steady state levels of the intermediates preceding the 3-phosphoglycerate kinase step were increased 2–3 fold. The subsequent metabolites were decreased or practically unmodified. ATP, ADP, NAD+, and NADH were not affected. The reduced glutathione level was increased and the ratio of reduced to oxidized glutathione was doubled. The glycolytic output and its pH-dependency were normal. The metabolic significance of the enzyme defect was assessed by the in vitro creation of cell stressing conditions, i.e. low pH and high pyruvate levels. In both cases, the 3-phosphoglycerate kinase activity became limiting at low pH, glucose-6-phosphate accumulated at a faster rate and fructose- 1,6-diphosphate and dihydroxyacetone phosphate disappeared more slowly in the deficient cells. After pyruvate loading these cells showed: a faster, more pronounced rise in 1,3-diphosphoglycerate and a decrease in 2,3-diphosphoglycerate (slightly increased in the controls): a drop in reduced glutathione (constant in the controls): constant ATP and slightly increased 3-phosphoglycerate concentrations (both strongly increased in the controls): a slight increase in NADH (dropped to nil in the controls). Steady state glycolysis under normal conditions seemed to be affected by the enzyme deficiency. 3-phosphoglycerate kinase however, became more severely limiting a low pH or after the addition of pyruvate. In these conditions, the flow was diverted to the 2,3-diphosphoglycerate bypass, less ATP was produced and the concentration of reduced glutathione decreased. This may be assumed to have led to impairment of the ionic pump and may thus explain the increased haemolysis.