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ABSTRACT: Recently, we reported a new mutation of phosphoglycerate kinase (PGK), called PGK-Barcelona, which causes chronic hemolytic anemia associated with progressive neurological impairment. We found a 140T→A substitution that produces an Ile46Asn change located at the N-domain of the enzyme and we suggested that the decrease of the PGK activity is probably related to a loss of enzyme stability. In this paper, by analyzing whole hemolysates and cloned enzymes, we show that both enzymes possess similar kinetic properties (although some differences are observed in the Km values) and the same electrophoretic mobility. However, PGK-Barcelona has higher thermal instability. Therefore, we confirm that the decrease of the red blood cell (RBC) PGK activity caused by the PGK-Barcelona mutation is more closely related to a loss of enzyme stability than to a decrease of enzyme catalytic function. Furthermore, we have measured the levels of glycolytic metabolites and adenine nucleotides in the RBC from controls and from the patient. The increase of 2,3-bisphosphoglycerate and the decrease of ATP RBC levels are the only detected metabolic changes that could cause hemolytic anemia.
Blood Cells Molecules and Diseases 01/2011; 46(3):206-11. · 2.35 Impact Factor
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ABSTRACT: Molecular characteristics of red blood cell (RBC) glucose phosphate isomerase (GPI) deficiency are described in two Spanish patients with chronic nonspherocytic hemolytic anemia. One patient, with residual GPI activity in RBCs of around 7% (GPI-Catalonia), is homozygous for the missense mutation c.1648A>G (p.Lys550Glu) in exon 18. The other patient, with residual activity in RBCs of around 20% (GPI-Barcelona), was found to be a compound heterozygote for two different missense mutations: c.341A>T (p.Asp113Val) in exon 4 and c.663T>G (p.Asn220Lys) in exon 7. Molecular modeling using the human crystal structure of GPI as a model was performed to determine how these mutations could affect enzyme structure and function.
Human Mutation 12/2006; 27(11):1159. · 5.69 Impact Factor
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ABSTRACT: In a patient with clinical diagnosis of Hereditary Spherocytosis and partial deficiency (50%) of red blood cell phosphoglycerate mutase (PGAM) activity, we have recently reported [A. Repiso, P. Pérez de la Ossa, X. Avilés, B. Oliva, J. Juncá, R. Oliva, E. Garcia, J.L.L. Vives-Corrons, J. Carreras, F. Climent, Red blood cell phosphoglycerate mutase. Description of the first human BB isoenzyme mutation, Haematologica 88 (2003) (03) ECR07] the first described mutation of type B PGAM subunit that as a dimer constitutes the PGAM (EC 5.4.2.1) isoenzyme present in red blood cells. The mutation is the substitution c.690G>A (p.Met230Ile). In this report, we show that the mutated PGAM possesses an abnormal behaviour on ion-exchange chromatography and is more thermo-labile that the native enzyme. We also confirm that, similar to the PGAM isoenzymes from other sources, the BB-PGAM from human erythrocytes has a ping pong or phosphoenzyme mechanism, and that the mutation does not significantly change the K(m) and K(i) values, and the optimum pH of the enzyme. The increased instability of the mutated enzyme can account for the decreased PGAM activity in patient's red blood cells. However, the implication of a change of the k(cat) produced by the mutation cannot be discarded, since we could not determine the k(cat) value of the mutated PGAM.
Biochimica et Biophysica Acta 06/2005; 1740(3):403-10. · 4.66 Impact Factor
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ABSTRACT: We previously reported the first case of red blood cell phosphoglycerate mutase (PGAM) isozyme BB deficiency due to the homozygous point mutation cDNA 690G->A, which causes a substitution of methionine 230 by isoleucine. In the present work we analyzed the changes in glycolytic intermediates caused by this mutation. With the exception of hexose phosphates, all other intermediates were decreased. In contrast, lactate levels were increased. The methionine 230 isoleucine change did not alter the mutated PGAM levels.
Haematologica 03/2005; 90(2):257-9. · 6.42 Impact Factor
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ABSTRACT: The effects of triiodothyronine (T(3)) and hypoxia on 2,3-bisphosphoglycerate (2,3-BPG) studied in vitro are unclear. To clarify these effects we selected a more physiologic approach: the in vivo study in rabbits. We also present the changes produced by T(3) and hypoxia on phosphoglycerate mutase (PGAM), which requires 2,3-BPG as a cofactor, and 2,3-BPG synthase (BPGS), the enzyme responsible for 2,3-BPG synthesis in erythroblasts and reticulocytes.
Hyperthyroidism was induced by daily T(3) injection (250 microg/kg), hypoxia by a mixture of 90% nitrogen and 10% oxygen and hypothyroidism by propylthiouracil (PTU) added to drinking water.
Both T(3) administration and hypoxic conditions increased 2,3-BPG levels and BPGS mRNA levels and activity in erythroblasts but not in reticulocytes. Unlike BPGS, both PGAM mRNA levels and activity were increased in erythroblasts and reticulocytes under hyperthyrodism and hypoxia. The antihormone PTU produced opposite effects to T(3).
The results presented here suggest that both hyperthyroidism and hypoxia modulate in vivo red cell 2,3-BPG content by changes in the expression of BPGS. Similarly, the changes in PGAM activity are also explained by changes in its expression.
Hormone Research 02/2004; 62(4):191-6. · 2.48 Impact Factor
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Haematologica 04/2003; 88(3):ECR07. · 6.42 Impact Factor
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ABSTRACT: In the present work, we studied the effects of hypoxia and triiodothyronine (T(3)) on phosphoglycerate mutase (PGAM) activity and expression in rabbit liver, brain, and skeletal muscle under in vivo conditions.
Hypoxia was induced in a methacrylate cage with a mixture of 90% nitrogen and 10% oxygen. Hyperthyroidism was induced daily by T(3) injection (250 microg/kg).
Hypoxia increases the PGAM activity in liver and brain, tissues which possess type PGAM-BB isozyme, but does not affect the PGAM activity in muscle which possesses type PGAM-MM isozyme. T(3) administration increases the PGAM activity in muscle and liver, but does not affect the enzyme activity in the brain. In all cases, the activity changes in parallel with those of PGAM mRNA levels.
The tissue-specific effects of hypoxia and T(3) could be explained by the tissue-specific distribution of both PGAM isozyme and T(3) receptors.
Hormone Research 02/2003; 59(1):16-20. · 2.48 Impact Factor
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Haematologica 05/2002; 87(4):ECR12. · 6.42 Impact Factor
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ABSTRACT: We previously showed that triiodothyronine (T3) stimulates muscle phosphoglycerate mutase (PGAM) activity and isozyme transition in rat skeletal and cardiac muscles.
The effects of T3 on PGAM types B and M subunit expression in rat muscle during development are reported.
T3 administration during the first 21 days of rat life more than doubles type M PGAM mRNA levels, but produces minor effects on type B PGAM mRNA levels. The antihormone propylthiouracil (PTU) slightly decreases both type B and M mRNA levels, but this decrease is not statistically significant.
Thyroid hormone influences PGAM mRNA isozyme levels differently and increases type M mRNA.
Hormone Research 02/2002; 57(1-2):48-52. · 2.48 Impact Factor
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ABSTRACT: 1.1. Phosphofructo 2-kinase from chicken erythrocytes copurifies with fructose 2,6-bisphosphatase activity, suggesting that the enzyme is bifunctional.2.2. Similarly to phosphofructo 2-kinase from other tissues it is activated by inorganic phosphate, and inhibited by phosphoenol pyruvate, sn-glycerol 3-phosphate and citrate. However, it has some characteristics different than those of chicken liver phosphofructo 2-kinase, indicating that it is a distinct isozyme.3.3. The phosphofructo 2-kinase/fructose 2,6-bisphosphatase activity ratio of the erythrocyte enzyme is one order of magnitude higher than of the enzyme from liver. In contrast with the chicken liver enzyme, phosphofructo 2-kinase from chicken erythrocytes is activated by dithiothreitol and its activity increases with pH.4.4. Chicken erythrocyte phosphofructo 2-kinase activity is neither modified by cyclic AMP-dependent protein kinase or casein kinase I and II. In contrast, it is partially inhibited by protein kinase C.
Comparative biochemistry and physiology. B, Comparative biochemistry 02/1990;
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ABSTRACT: 2,3-Bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) was purified and characterized from maize. SDS electrophoresis showed only one band with a molecular mass of 64 kDa, similar to that determined for the native enzyme by gel-filtration chromatography. The kinetic constants were similar to those reported for wheat germ phosphoglycerate mutase. Rabbit antiserum against maize phosphoglycerate mutase possesses a high degree of specificity. It also reacts with the wheat germ enzyme but fails to react with other cofactor-independent or cofactor-dependent phosphoglycerate mutases. Cell-free synthesis experiments indicate that phosphoglycerate mutase from maize is not post-translationally modified.
European Journal of Biochemistry. 11/1989; 186(1‐2):149 - 153.
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ABSTRACT: 1.1. Phosphofructokinase (EC 2.7.1.11) from chicken erythrocytes is activated by fructose 2,6-bisphosphate, glucose 1,6-bisphosphate and AMP, and it is inhibited by 2,3-bisphosphoglycerate and inositol hexaphosphate.2.2. The stimulatory effects produced by the two bisphosphorylated hexoses are additive and the effects produced by fructose 2,6-bisphosphate and by AMP are synergistic.3.3. The activatory effect produced by fructose 2,6-bisphosphate is counteracted by fructose 1,6-bisphosphate.4.4. The inhibition produced by both 2,3-bisphosphoglycerate and inositol hexaphosphate is released by fructose 2,6-bisphosphate.5.5. It is concluded that, like phosphofructokinase from mammalian tissues, the enzyme from chicken erythrocytes can be modulated by the relative concentrations of those metabolites.
Comparative biochemistry and physiology. B, Comparative biochemistry 02/1988;
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ABSTRACT: 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/109 cells; 238 ± 81 Uμ/109 cells) than in reticulocytes (40 ± 15 pmol/109 cells; 11 ± 3 Uμ/109 cells) and much higher than in mature erythrocytes (10 ± 0.8 pmol/109 cells 2 ± 1 Uμ/109 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.
FEBS Letters 10/1987; · 3.54 Impact Factor
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ABSTRACT: 1.1. The levels of glycerate 2,3-P2 and of 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities have been determined in isolated rat hepatocytes and adipocytes and in perfused rat tissues to discard blood contamination.2.2. The values obtained are much lower than those previously reported, ranging 0.50–40 nmol/g tissue.3.3. No relationship appears to exist between glycerate 2,3-P2 concentration and the levels of the enzymatic activities involved in glycerate 2,3-P2 metabolism.4.4. Assay of glycerate 2,3-P2 in tissue extracts constitute a very useful way to quantify blood contamination.
Comparative biochemistry and physiology. B, Comparative biochemistry 02/1987;
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ABSTRACT: In contrast to mammalian erythrocytes, chicken erythrocytes contain fructose 2,6-bisphosphate at levels (0.5 cells) similar to those of 2,3-bisphosphoglycerate (1.2 nmol/109 cells) and slightly lower than those of glucose 1,6-bisphosphate (5.2 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.
FEBS Letters 01/1987; · 3.54 Impact Factor
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ABSTRACT: 2,3-Bisphosphoglycerate, glucose 1,6-P, and fructose 2,6-P2 have been recognized as regulatory signals implicated in the control of metabolism, oxygen affinity of red cells and other cellular functions. The alterations of their metabolism constitute a novel area in molecular pathology. The concentration of 2,3-bisphosphoglycerate in erythrocytes changes in a number of pathological conditions. An inherited deficiency of the multifunctional enzyme involved in the synthesis and breakdown of 2,3-bisphosphoglycerate in erythrocytes has been reported. The levels of glucose 1,6-P2 are reduced in the liver and in the muscle of rats with experimentally induced diabetes. In muscle of genetically dystrophic mice a decrease in the levels of glucose 1,6-P2 has been found, probably resulting from enhancement of glucose 1,6-P2 phosphatase activity. Fructose 2,6-P2 levels are decreased in the liver of experimental diabetic mice and rats, and elevated in the liver of genetically obese animals.
Clinical Biochemistry 01/1987; · 2.08 Impact Factor
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ABSTRACT: Fructose 2,6-bisphosphate affects phosphoglucomutase from plant and animal sources in a similar way. As previously found with rabbit muscle phosphoglucomutase, fructose 2,6-bisphosphate cannot substitute for glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by phosphoglucomutase from potato tubers, pea seeds, and string-beans. In the presence of glucose 1,6-bisphosphate, fructose 2,6-bisphosphate inhibits phosphoglucomutase from potato tubers. Activation of phosphoglucomutase from plant sources by fructose 2,6-bisphosphate reported by others was probably due to contamination of the commercial preparation of fructose 2,6-bisphosphate by glucose 1,6-bisphosphate.
Plant physiology 11/1986; 82(2):619-21. · 6.53 Impact Factor
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ABSTRACT: Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phosph glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P2 phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.
Biochemical and Biophysical Research Communications 08/1981; · 2.48 Impact Factor
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ABSTRACT: 1.1. Glycerate 1,3-P2-dependent glucose, 1,6-P2 synthase has been purified 2000-fold from pig skeletal muscle, with a yield of 75%.2.2. The enzyme possesses fructose 1,6-P2-dependent glucose 1,6-P2 synthase and phosphoglucomutase activities, which represent 0.1 and 60% of the main activity, respectively.3.3. Both glucose 1-P and glucose 6-P can act as acceptors of the phosphoryl group from glycerate 1,3-P2.4.4. The values are 19 μM and 67 nM for glucose 1-P and glycerate 1,3-P2, respectively.5.5. The enzyme is inhibited by glycerate 2,3-P2, fructose 1,6-P2, glycerate 3-P, phosphoenolpyruvate and lithium, the inhibition pattern varying with the compound.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry.
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ABSTRACT: 1.1. Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose ; (B) glucose ; (C) glucose ; (D) glucose .2.2. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis.3.3. With the exception of skeletal muscle, activity “D” represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity “B” is the major activity.4.4. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction “D” is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography.5.5. There is an enzyme which specifically catalyzes reaction “C”, not previously described.6.6. There is a glucose 1-P kinase not identical to phosphofructokinase.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry.
