RESUMEN
PURPOSE: Oxygen is vital for oocyte maturation; however, oxygen regulation within ovarian follicles is not fully understood. Hemoglobin is abundant within the in vivo matured oocyte, indicating potential function as an oxygen regulator. However, hemoglobin is significantly reduced following in vitro maturation (IVM). The molecule 2,3-bisphosphoglycerate (2,3-BPG) is essential in red blood cells, facilitating release of oxygen from hemoglobin. Towards understanding the role of 2,3-BPG in the oocyte, we characterized gene expression and protein abundance of bisphosphoglycerate mutase (Bpgm), which synthesizes 2,3-BPG, and whether this is altered under low oxygen or hemoglobin addition during IVM. METHODS: Hemoglobin and Bpgm expression within in vivo matured human cumulus cells and mouse cumulus-oocyte complexes (COCs) were evaluated to determine physiological levels of Bpgm. During IVM, Bpgm gene expression and protein abundance were analyzed in the presence or absence of low oxygen (2% and 5% oxygen) or exogenous hemoglobin. RESULTS: The expression of Bpgm was significantly lower than hemoglobin when mouse COCs were matured in vivo. Following IVM at 20% oxygen, Bpgm gene expression and protein abundance were significantly higher compared to in vivo. At 2% oxygen, Bpgm was significantly higher compared to 20% oxygen, while exogenous hemoglobin resulted in significantly lower Bpgm in the COC. CONCLUSION: Hemoglobin and 2,3-BPG may play a role within the maturing COC. This study shows that IVM increases Bpgm within COCs compared to in vivo. Decreasing oxygen concentration and the addition of hemoglobin altered Bpgm, albeit not to levels observed in vivo.
Asunto(s)
Bisfosfoglicerato Mutasa/genética , Técnicas de Maduración In Vitro de los Oocitos , Oocitos/crecimiento & desarrollo , Oogénesis/genética , 2,3-Difosfoglicerato/sangre , Animales , Bisfosfoglicerato Mutasa/sangre , Blastocisto/metabolismo , Células del Cúmulo , Femenino , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Meiosis/genética , Ratones , Folículo Ovárico/crecimiento & desarrolloRESUMEN
BACKGROUND: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. METHODS: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. RESULTS: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. CONCLUSIONS: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.
Asunto(s)
Proteínas Quinasas Activadas por AMP/sangre , Adaptación Fisiológica/fisiología , Mal de Altura/sangre , Eritrocitos/metabolismo , Receptor de Adenosina A2B/sangre , 2,3-Difosfoglicerato/sangre , 5'-Nucleotidasa/sangre , 5'-Nucleotidasa/deficiencia , Lesión Pulmonar Aguda/fisiopatología , Adenosina/sangre , Adulto , Mal de Altura/enzimología , Mal de Altura/fisiopatología , Animales , Bisfosfoglicerato Mutasa/sangre , Activación Enzimática , Proteínas Ligadas a GPI/sangre , Humanos , Metaboloma , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxígeno/sangre , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
The trifunctional enzyme bisphosphoglyceromutase (or diphosphoglycerate mutase) (EC 2.7.5.4) was purified from human red cells and injected into two chickens. Specific anti-bisphosphoglyceromutase antibodies were produced that displayed a single precipitation line on Ouchterlony plates and on immunoelectrophoresis. No cross-reaction of these antibodies was detected with phosphoglyceromutase, the common glycolytic enzyme. Immunoneutralization of bisphosphoglyceromutase and of its two other activities, i.e., bisphosphoglycerate phosphatase and phosphoglyceromutase, was observed for a purified preparation. The anti-bisphosphoglyceromutase antibody reacts with the inactive enzyme present in the hemolysate of a mutant human subject. It also binds bisphosphoglyceromutase inactivated by N-ethylmaleimide, a strong alkylating agent of SH groups. Active bisphosphoglyceromutase is stable at 55 degrees C, whereas the inactive forms of the mutant and of the alkylated hemolysates are thermolabile. These forms can be protected against thermal precipitation by 4 mM 2,3-diphosphoglycerate and 4 mM 3-phosphoglycerate. These findings afford evidence that the binding of the substrates on the bisphosphoglyceromutase molecule is not prevented by alkylation nor by the mutation of the hereditary inactive enzyme.
Asunto(s)
Bisfosfoglicerato Mutasa/genética , Eritrocitos/enzimología , Mutación , Fosfotransferasas/genética , Alquilación , Animales , Complejo Antígeno-Anticuerpo , Bisfosfoglicerato Mutasa/sangre , Bisfosfoglicerato Mutasa/aislamiento & purificación , Pollos/inmunología , Estabilidad de Medicamentos , Calor , Humanos , Inmunodifusión , Inmunoelectroforesis , CinéticaRESUMEN
Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.
Asunto(s)
Bisfosfoglicerato Mutasa/química , Eritrocitos/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Bisfosfoglicerato Mutasa/sangre , Catálisis , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Electrophoresis of 3-phosphoglycerate mutase from erythrocytes of man and several animal species has been performed on cellulose acetate strips. In most cases the electrophoretic pattern of this enzymatic activity shows three bands. 2,3-diphosphoglycerate phosphatase and diphosphoglycerate mutase from erythrocytes of the same species have been revealed after migration during the same electrophoresis. We found that the band of 2,3-diphosphoglycerate phosphatase and the band of diphosphoglycerate mutase activities migrate at the same level as one of the bands corresponding to 3-phosphoglycerate mutase. Here, we discuss the possible existence of a single molecule carrying three enzymatic activities.
Asunto(s)
Eritrocitos/enzimología , Fosfoglicerato Mutasa/sangre , Fosfotransferasas/sangre , Animales , Bisfosfoglicerato Mutasa/sangre , Ácidos Difosfoglicéricos , Electroforesis en Acetato de Celulosa , Cabras , Caballos , Humanos , Moluscos , Monoéster Fosfórico Hidrolasas/sangre , RatasRESUMEN
The fractionation of normal human erythrocytes by counter-current distribution (CCD) in charge-sensitive dextran-polyethylene glycol two-phase systems was confirmed and extended to red blood cells from heterozygous beta-thalassaemic patients. The differences between the distribution profiles of normal (homogeneous) and abnormal (heterogeneous) red blood cells reflect their different surface-charge properties. As suggested by the decline of membrane sialic acid released after neuraminidase treatment and the specific activities of two age-dependent enzymes (membrane acetylcholinesterase and intracellular pyruvate kinase) in the distribution profiles (from the left- to the right-hand side fractions), the fractionation seems to be according to red blood cell age. A constancy of the 2,3-bisphosphoglycerate level was observed in ageing red blood cells.
Asunto(s)
Separación Celular/métodos , Eritrocitos/citología , 2,3-Difosfoglicerato , Acetilcolinesterasa/sangre , Adulto , Bisfosfoglicerato Mutasa/sangre , Distribución en Contracorriente , Ácidos Difosfoglicéricos/sangre , Electroquímica , Envejecimiento Eritrocítico , Índices de Eritrocitos , Membrana Eritrocítica/química , Eritrocitos/química , Humanos , Ácido N-Acetilneuramínico , Neuraminidasa/farmacología , Piruvato Quinasa/sangre , Piruvatos/sangre , Ácido Pirúvico , Ácidos Siálicos/sangre , Agua , Talasemia beta/sangreRESUMEN
An increase in bisphosphoglycerate mutase (BPGM) and a decrease in pyruvate kinase (PK), i.e. a decrease in PK/BPGM ratio, was observed in red cell populations from anemic rats containing 95% down to 3% reticulocytes in blood. Such a ratio has been used to study the fractionation of recticulocytes, according to their degree of maturation, after counter-current distribution of those cell populations in dextranpoly (ethylene glycol) two-phase systems. When applying this procedure to the fractionation according to age of erythrocytes from normal rats, the decrease of PK with cellular age was observed without a significant variation in BPGM activity.
Asunto(s)
Bisfosfoglicerato Mutasa/sangre , Envejecimiento Eritrocítico , Eritrocitos/enzimología , Fosfotransferasas/sangre , Piruvato Quinasa/sangre , Reticulocitos/enzimología , Anemia/enzimología , Animales , Eritropoyesis , RatasRESUMEN
The specific activity of several red cell enzymes was studied in a pair of monozygotic twins, one of whom presented acute myeloblastic leukemia. When the intrapair variation of these twins was compared with that of a series of nine normal twins, a significant decrease in phosphoglycerate kinase, diphosphoglycerate mutase, pyruvate kinase, lactic dehydrogenase, adenylate kinase and glucose-6-phosphate dehydrogenase activities and an increase in 6-phosphogluconate dehydrogenase activity were demonstrable for the leukemic twin. The heat stability of the leukemic proband's pyruvate kinase at pH 8.0 and 56 degrees C was less than that of the normal twin, suggesting an acquired qualitative disorder.
Asunto(s)
Enfermedades en Gemelos , Eritrocitos/enzimología , Leucemia Mieloide Aguda/enzimología , Piruvato Quinasa/sangre , Adenilil Ciclasas/sangre , Adulto , Bisfosfoglicerato Mutasa/sangre , Femenino , Glucosafosfato Deshidrogenasa/sangre , Humanos , L-Lactato Deshidrogenasa/sangre , Leucemia Mieloide Aguda/genética , Fosfoglicerato Quinasa/sangre , Gemelos MonocigóticosRESUMEN
A mathematical model for the glycolysis of human erythrocytes is presented which takes into account ATP-synthesis and -consumption. A set of three differential equations describes the steady states and the time-dependent changes of the metabolite concentrations under blood storage conditions. For a given parameter combination there are in general three stationary points of the system, one of which is unstable. At a low ATP-need the ATP-level is relatively constant for variations in the rate constant of the ATP-consuming processes. Above a critical level of the energy consumption the system breaks down. An important role of the 2.3P2G-bypass of the erythrocytes is its action as an "energy buffer", wasting ATP in case of ATP-overproduction and producing ATP in case of underproduction. A parameter combination consistent with the data on the isolated enzymes was found which gives a good agreement of theoretical predictions with the measured metabolite concentrations. Under blood preservation conditions the difference of the rates of ATP-production and -consumption is the most important factor for a high ATP-level over long periods.
Asunto(s)
Eritrocitos/enzimología , Glucólisis , Adenosina Trifosfatasas/sangre , Bisfosfoglicerato Mutasa/sangre , Ácidos Difosfoglicéricos , Hexoquinasa/sangre , Humanos , Cinética , Matemática , Modelos Biológicos , Fosfofructoquinasa-1/sangre , Monoéster Fosfórico Hidrolasas/sangre , Piruvato Quinasa/sangreRESUMEN
The age changes of 2,3-DPG concentration, 2,3-diphosphoglycerate mutase, hexokinase and pyruvate kinase activities in pigs erythroid cells during the first 10 days after birth have been investigated for study of the mechanism of blood oxygen-transport function regulation at the early stages of postnatal adaptation. It was established, that there are age peculiarities in young, mature and old erythrocytes metabolism in piglets during the transition to postnatal development. It was shown that the rise of 2,3-DPG, the allosteric effector of hemoglobin oxygen affinity level, is due to the increased activities of 2,3-diphosphoglycerate mutase and hexokinase after birth, that is particularly characteristic of the young pigs erythrocytes.
Asunto(s)
Eritrocitos/enzimología , Glucólisis/fisiología , 2,3-Difosfoglicerato , Adaptación Fisiológica , Animales , Animales Recién Nacidos , Transporte Biológico/fisiología , Bisfosfoglicerato Mutasa/sangre , Ácidos Difosfoglicéricos/sangre , Metabolismo Energético/fisiología , Hexoquinasa/sangre , Piruvato Quinasa/sangre , PorcinosAsunto(s)
Bisfosfoglicerato Mutasa/sangre , Eritrocitos/enzimología , Fosfotransferasas/sangre , Hormonas Tiroideas/farmacología , Interacciones Farmacológicas , Glicerofosfatos/farmacología , Humanos , Técnicas In Vitro , Cinética , Nicotina/farmacología , Tiroxina/farmacología , Factores de Tiempo , Triyodotironina/farmacologíaAsunto(s)
Bisfosfoglicerato Mutasa/sangre , Carbohidrato Epimerasas/sangre , Electroforesis en Gel de Almidón/métodos , Nucleotidiltransferasas/sangre , Pentosiltransferasa/sangre , Fosfotransferasas/sangre , Purina-Nucleósido Fosforilasa/sangre , Triosa-Fosfato Isomerasa/sangre , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/sangre , Eritrocitos/enzimología , HumanosAsunto(s)
Anemia Hemolítica Congénita/enzimología , Errores Innatos del Metabolismo/complicaciones , Adulto , Anemia Hemolítica Congénita/complicaciones , Bisfosfoglicerato Mutasa/sangre , Checoslovaquia , Femenino , Deficiencia de Glucosafosfato Deshidrogenasa/complicaciones , Humanos , Masculino , Errores Innatos del Metabolismo/enzimologíaRESUMEN
New cases of diphosphoglyceromutase (DPGM) have been detected, associated with erythrocytosis in two unrelated families. The deficiency appears to be inherited as an autosomal dominant trait. Diphosphoglycerate phosphatase activity paralleled DPGM activity in all the subjects. Three of the latter displayed complete DPGM deficiency with about 0.4% of the normal 2,3-diphosphoglycerate (2,3 DPG) level. The other four showed partial deficiency (about 50% normal mean) with a similar decrease in 2,3-DPG level. The P50 values are in agreement with the red cell 2,3-DPG concentrations. Il all the deficient subjects the ATP level was elevated and the pattern of glycolytic intermediates was disturbed, with an increase in fructose 1,6-diphosphate, triose-phosphates, 3-phosphoglycerate, glucose 1,6-diphosphate, and reduced or normal levels of glucose-6-phosphate and fructose-6-phosphate.
Asunto(s)
Bisfosfoglicerato Mutasa/deficiencia , Fosfotransferasas/deficiencia , Policitemia/genética , Adulto , Bisfosfoglicerato Mutasa/sangre , Preescolar , Femenino , Glucólisis , Humanos , Masculino , Persona de Mediana Edad , Linaje , Policitemia/sangreRESUMEN
Two generations of red cells (embryonic and definitive), different types of haemoglobins, and special organic phosphates involved in the control of haemoglobin oxygenation (2:3-bisphosphoglycerate, BPG, and inositol-5-phosphate, IP5), have been found progressively during development of the chick. Levels of both organic phosphates, as well as activities of the enzymes involved in BPG synthesis (2:3-bisphosphoglycerate synthase, BPGM) and IP5 formation (phytase), were studied in the erythrocyte populations from embryo, young and adult chickens. Measurement of specific activities of BPGM and phytase in the two subpopulations present in young chickens showed that these phosphates could be specifically and predominantly formed in these two red cell populations.
Asunto(s)
6-Fitasa/sangre , Bisfosfoglicerato Mutasa/sangre , Ácidos Difosfoglicéricos/sangre , Eritropoyesis , Fosfatos de Inositol/sangre , Fosfotransferasas/sangre , Fosfatos de Azúcar/sangre , Factores de Edad , Animales , Embrión de PolloRESUMEN
Diphosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes. The native enzyme has a molecular weight of 57 000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of about 26 500, indicating that diphosphoglycerate mutase is comprised of two subunits of similar mass. The enzyme exhibits the following intrinsic activities: diphosphoglyceratemutase, monophosphoglycerate mutase, and 2,3-diphosphoglycerate phosphatase. The latter activity is enhanced in the presence of either organic or inorganic anions. Glycolate-2-P, particularly, has a profound activating effect. Nonspecific phosphatase and enolase activities are absent. The enzyme has an extinction coefficient at 280 nm of 1.65 cm2/mg. The amino acid composition of the homogeneous protein has been determined.
Asunto(s)
Bisfosfoglicerato Mutasa/sangre , Eritrocitos/enzimología , Fosfotransferasas/sangre , Aminoácidos/análisis , Bisfosfoglicerato Mutasa/aislamiento & purificación , Hemólisis , Calor , Humanos , Peso MolecularRESUMEN
Rare genetic variants of human red cell 2,3-diphosphoglycerate mutase (DPGM) and monophosphoglycerate mutase (MPGM) were compared by starch gel electrophoresis. The isozyme patterns showed that genetic variation of the enzymes were independent from each other, thus DPGM and MPGM must be controlled by two separate loci.