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1.
J Assist Reprod Genet ; 38(6): 1363-1372, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34052998

RESUMO

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.


Assuntos
Bisfosfoglicerato Mutase/genética , Técnicas de Maturação in Vitro de Oócitos , Oócitos/crescimento & desenvolvimento , Oogênese/genética , 2,3-Difosfoglicerato/sangue , Animais , Bisfosfoglicerato Mutase/sangue , Blastocisto/metabolismo , Células do Cúmulo , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Meiose/genética , Camundongos , Folículo Ovariano/crescimento & desenvolvimento
2.
Circulation ; 134(5): 405-21, 2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27482003

RESUMO

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.


Assuntos
Proteínas Quinases Ativadas por AMP/sangue , Adaptação Fisiológica/fisiologia , Doença da Altitude/sangue , Eritrócitos/metabolismo , Receptor A2B de Adenosina/sangue , 2,3-Difosfoglicerato/sangue , 5'-Nucleotidase/sangue , 5'-Nucleotidase/deficiência , Lesão Pulmonar Aguda/fisiopatologia , Adenosina/sangue , Adulto , Doença da Altitude/enzimologia , Doença da Altitude/fisiopatologia , Animais , Bisfosfoglicerato Mutase/sangue , Ativação Enzimática , Proteínas Ligadas por GPI/sangue , Humanos , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/sangue , Fosforilação , Processamento de Proteína Pós-Traducional
3.
Horm Res ; 62(4): 191-6, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15375329

RESUMO

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.


Assuntos
2,3-Difosfoglicerato/metabolismo , Bisfosfoglicerato Mutase/metabolismo , Eritroblastos/metabolismo , Hipóxia/metabolismo , Fosfoglicerato Mutase/metabolismo , Reticulócitos/metabolismo , Tri-Iodotironina/farmacologia , 2,3-Difosfoglicerato/sangue , Animais , Bisfosfoglicerato Mutase/sangue , Bisfosfoglicerato Mutase/genética , Hipertireoidismo/sangue , Hipertireoidismo/induzido quimicamente , Hipertireoidismo/metabolismo , Hipóxia/sangue , Masculino , Fosfoglicerato Mutase/sangue , Fosfoglicerato Mutase/genética , RNA Mensageiro/sangue , RNA Mensageiro/metabolismo , Coelhos
4.
Am J Hematol ; 75(4): 205-8, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15054810

RESUMO

A 28-year-old asymptomatic male of Iranian Jewish (Meshadi) heritage was found on routine exam to have an erythrocytosis (RBC = 6.22 x 10(12)/l, Hgb = 19.2 g/dl, Hct = 58.9%). Splenomegaly was absent on physical exam. There was no family history of erythrocytosis. His oxygen dissociation curve was left-shifted with a p50 of 19 mmHg (normal = 25-32 mmHg). Hemoglobin electrophoresis showed no abnormalities. DNA sequencing of the hemoglobin beta globin gene and both alpha globin genes did not reveal a mutation. A 2,3-bisphosphoglycerate (BPG) level was markedly decreased at 0.3 micromol/g Hb (normal = 11.4-19.4 micromol/g Hb). The patient's bisphosphoglycerate mutase (BPGM) enzyme activity was also markedly decreased at 0.16 IU/g Hb (normal = 4.13-5.43 IU/g Hb). A red cell enzyme panel revealed a markedly decreased G-6-PD level (0.3 U/g Hb, normal = 8.6-18.6 U/g Hb). His parents and a brother were also available for evaluation. Both parents showed normal 2,3-BPG levels but BPGM activity approximately 50% of normal. Paradoxically, the brother showed normal BPGM activity but a slightly decreased 2,3-BPG level. All family members had markedly decreased G-6-PD activity. DNA sequencing of the BPGM gene showed the propositus to be homozygous for 185 G-->A, Arg 62 Gln in exon 2. Thus, the erythrocytosis in this patient is secondary to low 2,3-BPG levels, due to a deficiency in BPG mutase. This appears due to consanguinity within this family.


Assuntos
Bisfosfoglicerato Mutase/deficiência , Deficiência de Glucosefosfato Desidrogenase/complicações , Policitemia/etiologia , Adulto , Bisfosfoglicerato Mutase/sangue , Bisfosfoglicerato Mutase/genética , Eritrócitos/metabolismo , Eritrócitos/patologia , Humanos , Masculino , Oxigênio/sangue , Pais , Policitemia/sangue , Policitemia/enzimologia , Policitemia/genética , Irmãos
6.
Nihon Rinsho ; 57 Suppl: 444-7, 1999 Aug.
Artigo em Japonês | MEDLINE | ID: mdl-10503466
7.
J Clin Lab Anal ; 12(5): 263-7, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9773955

RESUMO

We established an enzyme-linked immunosorbent assay (ELISA) system for the determination of human bisphosphoglycerate mutase (BPGM) protein content in human erythrocytes using a polyclonal anti-BPGM antibody, we determined reference ranges for BPGM protein content, synthase activity, and specific activity in human erythrocytes. We produced a recombinant human BPGM (rBPGM) by gene manipulation using E. coli and then obtained the polyclonal antibody by immunizing rabbits with purified rBPGM. The reproducibility of the ELISA was in an acceptable range with a coefficient of variation under 1.5%. The ELISA was reliable in the range of 0.1 to 10 ng/mL. The polyclonal anti-rBPGM antibody did not show any cross-reaction with recombinant human B type phosphoglycerate mutase, which is highly homologous to rBPGM. The ELISA was found to be practical for the determination of BPGM protein content in human erythrocytes. The mean BPGM protein content was 56.3+/-9.7 microg/mL in whole blood (mean+/-SD, n = 50). The ELISA can be used to examine various hematologic disorders with abnormal red cell size and cell counts, and to detect BPGM enzymopathy in human erythrocytes.


Assuntos
Bisfosfoglicerato Mutase/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Eritrócitos/enzimologia , Adulto , Animais , Humanos , Coelhos , Proteínas Recombinantes/sangue , Valores de Referência , Reprodutibilidade dos Testes
9.
Ukr Biokhim Zh (1978) ; 66(5): 31-5, 1994.
Artigo em Ucraniano | MEDLINE | ID: mdl-7747343

RESUMO

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.


Assuntos
Eritrócitos/enzimologia , Glicólise/fisiologia , 2,3-Difosfoglicerato , Adaptação Fisiológica , Animais , Animais Recém-Nascidos , Transporte Biológico/fisiologia , Bisfosfoglicerato Mutase/sangue , Ácidos Difosfoglicéricos/sangue , Metabolismo Energético/fisiologia , Hexoquinase/sangue , Piruvato Quinase/sangue , Suínos
10.
J Chromatogr A ; 668(1): 165-71, 1994 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-8004229

RESUMO

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.


Assuntos
Separação Celular/métodos , Eritrócitos/citologia , 2,3-Difosfoglicerato , Acetilcolinesterase/sangue , Adulto , Bisfosfoglicerato Mutase/sangue , Distribuição Contracorrente , Ácidos Difosfoglicéricos/sangue , Eletroquímica , Envelhecimento Eritrocítico , Índices de Eritrócitos , Membrana Eritrocítica/química , Eritrócitos/química , Humanos , Ácido N-Acetilneuramínico , Neuraminidase/farmacologia , Piruvato Quinase/sangue , Piruvatos/sangue , Ácido Pirúvico , Ácidos Siálicos/sangue , Água , Talassemia beta/sangue
11.
Biochimie ; 74(6): 519-26, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1387804

RESUMO

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.


Assuntos
Bisfosfoglicerato Mutase/química , Eritrócitos/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Bisfosfoglicerato Mutase/sangue , Catálise , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Relação Estrutura-Atividade , Difração de Raios X
12.
Biochem J ; 279 ( Pt 1): 237-43, 1991 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-1656939

RESUMO

Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes.


Assuntos
Bisfosfoglicerato Mutase/sangue , Envelhecimento Eritrocítico , Eritrócitos/enzimologia , Fosfotransferases/metabolismo , Polímeros , Animais , Bisfosfoglicerato Mutase/isolamento & purificação , Distribuição Contracorrente , Glicólise , Humanos , Ratos , Ratos Endogâmicos , Água/química
13.
Mol Cell Biochem ; 99(1): 21-4, 1990 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-2177836

RESUMO

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.


Assuntos
Ácidos Difosfoglicéricos/sangue , Frutosedifosfatos/sangue , Glucose-6-Fosfato/análogos & derivados , Glucofosfatos/sangue , Reticulócitos/metabolismo , 2,3-Difosfoglicerato , Animais , Bisfosfoglicerato Mutase/sangue , Complexos Multienzimáticos , Fosfofrutoquinase-1/sangue , Monoéster Fosfórico Hidrolases/sangue , Piruvato Quinase/sangue , Ovinos
14.
J Biol Chem ; 264(32): 18966-72, 1989 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-2553728

RESUMO

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.


Assuntos
Bisfosfoglicerato Mutase/genética , Escherichia coli/genética , Mutação , Fosfotransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bisfosfoglicerato Mutase/sangue , Bisfosfoglicerato Mutase/metabolismo , Clonagem Molecular , Eritrócitos/enzimologia , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Vetores Genéticos , Humanos , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/síntese química , Plasmídeos , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
16.
Biochem Biophys Res Commun ; 153(3): 945-51, 1988 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-2839174

RESUMO

The effects of two sulfhydryl reagents on the three activities of bisphosphoglycerate mutase have been compared. Under N-ethylmaleimide treatment all the activities were inhibited except for 60% of the non-stimulated phosphatase. With iodoacetamide the mutase and the stimulated-phosphatase activities were completely inhibited whereas the non-stimulated phosphatase and 60% of the synthase activities were unaffected. 2,3-bisphosphoglycerate protected all the activities of the enzyme against inactivation by the two sulfhydryl reagents whereas 3-phosphoglycerate protected them only against iodoacetamide. 2-phosphoglycolate had an identical effect to that of 3-phosphoglycerate except for its effect on the non-stimulated phosphatase activity, which was slightly enhanced under N-ethylmaleimide treatment.


Assuntos
Bisfosfoglicerato Mutase/sangue , Eritrócitos/enzimologia , Etilmaleimida/farmacologia , Iodoacetamida/farmacologia , Iodoacetatos/farmacologia , Fosfotransferases/sangue , 2,3-Difosfoglicerato , Ácidos Difosfoglicéricos/metabolismo , Ácidos Glicéricos/metabolismo , Humanos
17.
Biosci Rep ; 7(2): 113-9, 1987 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2820524

RESUMO

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.


Assuntos
Bisfosfoglicerato Mutase/sangue , Envelhecimento Eritrocítico , Eritrócitos/enzimologia , Fosfotransferases/sangue , Piruvato Quinase/sangue , Reticulócitos/enzimologia , Anemia/enzimologia , Animais , Eritropoese , Ratos
18.
Biomed Biochim Acta ; 46(2-3): S126-30, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3036106

RESUMO

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate (DPG), the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA either by conventional cloning methods in plasmid pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated was 1673 bases, and encodes for a protein of 258 amino acids; it contains a large 3' untranslated region (785 bases). It is slightly smaller than the size of the intact mRNA estimated by Northern blot (1800 bases). Our sequence data indicate differences with the previously published amino acid sequence involving 21% of the residues. They were entirely confirmed by the amino acid composition of the tryptic peptides derived from purified BPGM. The revised amino acid sequence of the human BPGM is presented.


Assuntos
Bisfosfoglicerato Mutase/genética , DNA/genética , Fosfotransferases/genética , Sequência de Aminoácidos , Bisfosfoglicerato Mutase/sangue , Clonagem Molecular , Humanos , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reticulócitos/enzimologia
19.
EMBO J ; 5(9): 2275-83, 1986 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3023066

RESUMO

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.


Assuntos
Bisfosfoglicerato Mutase/sangue , Clonagem Molecular , DNA/metabolismo , Eritrócitos/enzimologia , Fosfotransferases/sangue , Sequência de Aminoácidos , Sequência de Bases , Bisfosfoglicerato Mutase/genética , Bisfosfoglicerato Mutase/isolamento & purificação , Enzimas de Restrição do DNA , Humanos , Peso Molecular , Plasmídeos
20.
Braz J Med Biol Res ; 19(1): 63-7, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-2948602

RESUMO

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.


Assuntos
Doenças em Gêmeos , Eritrócitos/enzimologia , Leucemia Mieloide Aguda/enzimologia , Piruvato Quinase/sangue , Adenilil Ciclases/sangue , Adulto , Bisfosfoglicerato Mutase/sangue , Feminino , Glucosefosfato Desidrogenase/sangue , Humanos , L-Lactato Desidrogenase/sangue , Leucemia Mieloide Aguda/genética , Fosfoglicerato Quinase/sangue , Gêmeos Monozigóticos
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