RESUMO
PURPOSE: To demonstrate the usefulness of biologic material obtained from distal embolic protection devices (DEPDs) used in carotid angioplasty for the study of atherosclerosis protein markers and to establish the effect of systemic inflammation on the protein expression of carotid atheromatous plaques. MATERIALS AND METHODS: Two-dimensional gel electrophoresis and mass spectrometry were used to study proteins obtained from debris captured in DEPDs from patients who underwent carotid angioplasty. In addition, protein expression obtained from angioplasty samples in patients with different types of systemic inflammation (measured by serum levels of high-sensitivity C-reactive protein [CRP] with a cutoff value of 3 mg/L) was compared. Finally, immunohistochemistry of atherosclerotic plaques obtained by endarterectomy was used to validate the results obtained using DEPDs. RESULTS: Proteomic studies were successfully performed using debris from DEPDs. Protein expression differences were found in debris from patients with high systemic inflammation compared with debris from patients with low systemic inflammation. Annexin A5 (ANXA5), haptoglobin precursor, purine nucleoside phosphorylase, transgelin-2 (TAGLN2), and bisphosphoglycerate mutase were upregulated in debris from patients with high systemic inflammation, and proteasome subunit 8 beta type and glutathione-S-transferase kappa 1 (GSTK1) levels were higher in debris from patients with low levels of systemic inflammation. CONCLUSIONS: Atherosclerotic plaque debris captured in DEPDs is a suitable and valid source of material for proteomic studies of atherosclerosis. Protein expression in DEPD debris is affected by systemic inflammation.
Assuntos
Angioplastia/instrumentação , Artérias Carótidas/química , Estenose das Carótidas/terapia , Dispositivos de Proteção Embólica , Proteínas/análise , Proteômica , Idoso , Anexina A5/análise , Biomarcadores/análise , Bisfosfoglicerato Mutase/análise , Proteína C-Reativa/análise , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Estenose das Carótidas/metabolismo , Estenose das Carótidas/patologia , Estenose das Carótidas/cirurgia , Distribuição de Qui-Quadrado , Eletroforese em Gel Bidimensional , Endarterectomia das Carótidas , Feminino , Glutationa Transferase/análise , Humanos , Imuno-Histoquímica , Inflamação/sangue , Inflamação/patologia , Mediadores da Inflamação/sangue , Masculino , Espectrometria de Massas , Proteínas dos Microfilamentos/análise , Pessoa de Meia-Idade , Proteínas Musculares/análise , Placa Aterosclerótica , Complexo de Endopeptidases do Proteassoma/análise , Proteômica/métodos , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Espanha , StentsRESUMO
2,3-Bisphosphoglycerate mutase (2,3-BPGM), an erythroid-expressed enzyme, synthesises 2,3-bisphosphoglycerate (2,3-BPG), the allosteric modulator of haemoglobin. This ligand has a higher affinity for adult haemoglobin than for fetal haemoglobin and differential binding of it facilitates transfer of oxygen between adult and fetal blood by lowering the affinity of adult haemoglobin for oxygen. This paper reports the discovery that 2,3-BPGM is synthesised in non-erythroid cells of the human placenta. Western blot analysis of placental extracts revealed high levels of 2,3-BPGM in the human placenta. Immunohistochemical staining and in situ hybridisation experiments indicated that abundant 2,3-BPGM is present in the syncytiotrophoblast layer of the placental villi at the feto-maternal interface. A cytochemical staining technique showed that the placental 2,3-BPGM is active, indicating that 2,3-BPG is synthesised in the outermost cells of the placenta. These observations demonstrate an unexpected and abundant presence of an enzyme key to oxygen release from adult haemoglobin, at the interface between maternal and fetal circulations.
Assuntos
Bisfosfoglicerato Mutase/análise , Placenta/enzimologia , Bisfosfoglicerato Mutase/genética , Bisfosfoglicerato Mutase/metabolismo , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , RNA Mensageiro/análiseRESUMO
We have previously reported the possibility of modifying the electrical properties of cells by means of their interaction with a specific ligand carrying a polyelectrolyte (Anal Biochem 200: 280-285). This selective modification of receptor-containing cells changed their partition in a charge-sensitive aqueous two-phase system. We here present the fractionation of electrically modified erythroblasts by the use of an automatic multiple-partition procedure, counter-current distribution. The cells were fractionated according to the degree of differentiation of erythroblasts as evaluated from the hemoglobin content as well as the relative activities of the two enzymes, 3-phosphoglycerate kinase and bisphospho-glycerate mutase.
Assuntos
Separação Celular/métodos , Eritroblastos/química , Animais , Bisfosfoglicerato Mutase/análise , Diferenciação Celular , Distribuição Contracorrente , Eritroblastos/efeitos dos fármacos , Masculino , Fosfoglicerato Quinase/análise , Polilisina/farmacologia , Ratos , Ratos Wistar , Propriedades de Superfície/efeitos dos fármacos , Transferrina/farmacologiaRESUMO
Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.
Assuntos
Fermentação , Glicólise , Bactérias Anaeróbias Gram-Negativas/enzimologia , Álcool Desidrogenase/análise , Bisfosfoglicerato Mutase/análise , Hidrolases de Éster Carboxílico/análise , Gliceraldeído-3-Fosfato Desidrogenases/análise , Bactérias Anaeróbias Gram-Negativas/ultraestrutura , Hidroliases/análise , Isoenzimas/análise , Microscopia Imunoeletrônica , Oxirredutases/análise , Fosfoglicerato Mutase/análise , Fosfopiruvato Hidratase/análise , Piruvato Descarboxilase/análise , Piruvato Quinase/análise , Frações Subcelulares/enzimologia , Frações Subcelulares/ultraestruturaRESUMO
Counter-current distribution in non charge-sensitive aqueous poly(ethylene glycol)-dextran two phase systems allows the fractionation of rat bone marrow cells into two broad cell subpopulations with different distribution coefficients in a relatively short time. Morphological identification and enzymatic studies suggest that erythroid cells are mainly present in the subpopulation with the higher distribution coefficient. The distribution coefficient and, therefore, surface hydrophobicity of these cells, apparently increase in parallel with an increase in their degree of differentiation and maturation.
Assuntos
Células da Medula Óssea , Separação Celular/métodos , Eritrócitos , Reticulócitos , Animais , Bisfosfoglicerato Mutase/análise , Diferenciação Celular , Distribuição Contracorrente , Dextranos , Masculino , Polietilenoglicóis , Piruvato Quinase/análise , Ratos , Ratos WistarRESUMO
In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erythrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC2.7.5.4/EC3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.
Assuntos
Ácidos Difosfoglicéricos/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Reticulócitos/enzimologia , 2,3-Difosfoglicerato , Animais , Bisfosfoglicerato Mutase/análise , Diferenciação Celular , Indução Enzimática , Eritrócitos/enzimologia , Eritropoese , Masculino , Coelhos , Ratos , Ratos Endogâmicos , Reticulócitos/citologiaRESUMO
Phosphoglycerate mutase (PGM) and creatine phosphokinase (CK) occur as three isozymes (types MM, MB and BB) in mammals and these exhibit similar transitions during skeletal muscle development. To study the influence of innervation on this transition and on the maintenance of the isozyme phenotype in mature muscle, we have determined the changes produced by sciatic neurectomy in neonatal and adult rat hindlimb muscles. In 40-day-old rats, denervation decreased both PGM and CK activity, the effect being more pronounced in the fast-twitch extensorum digitorum longus (EDL) and gastrocnemius muscles than in the slow-twitch soleus muscle. It also produced a progressive increase in the proportion of MB- and BB-PGM isozymes in EDL and gastrocnemius but not in soleus, and an increase of MB- and BB-CK isozymes in all three muscles. In 5-day-old rats, denervation prevented the developmental increase of PGM and CK activity in all three muscles. Denervation also prevented the normal decrease in the relative amounts of the MB and BB isozymes of both enzymes which occur during postnatal muscle development. These results can be explained by the different effects of denervation upon slow and fast muscles, and by the distinct distribution of PGM and CK isozymes in rat type I and II muscle fibers.
Assuntos
Bisfosfoglicerato Mutase/análise , Creatina Quinase/análise , Denervação Muscular , Músculos/enzimologia , Fosfotransferases/análise , Animais , Animais Recém-Nascidos/metabolismo , Bisfosfoglicerato Mutase/metabolismo , Creatina Quinase/metabolismo , Membro Posterior , Isoenzimas , Masculino , Desenvolvimento Muscular , Músculos/metabolismo , Fenótipo , Ratos , Ratos EndogâmicosRESUMO
We have previously reported the isolation in pure form of the human erythrocyte phosphoglycerate mutase isozyme B. We now report the sequence of the whole protein and the identification of its N-terminal blocking group. The protein tryptic peptides of phosphoglycerate mutase isozyme B were isolated by high performance liquid chromatography and their sequence determined by microsequencing. The sequence and the nature of the blocking group of the N-terminal tryptic peptide was shown to be N-acetyl-Ala-Ala-Tyr-Lys by mass spectrometry. Overlaps of the tryptic peptides were obtained by studying the V8 Staphylococcus aureus protease peptides of the aminoethylated phosphoglycerate mutase isozyme B either by microsequencing or by mass spectrometry. The procedure used allowed us to obtain the sequence on a very small amount of material and in a short period of time. Our data agree well with those derived from the cDNA nucleotide sequence described by Sakoda et al. (Sakoda, S., Shanske, S., DiMauro, S., and Schon, E. A. (1988) J. Biol. Chem. 263, 16899-16905). In addition, our data directly indicate that the initiation codon does not introduce a methionine as N-terminal amino acid and allowed the identification of the acetyl N-terminal group.
Assuntos
Bisfosfoglicerato Mutase/análise , Eritrócitos/enzimologia , Fosfotransferases/análise , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/análiseRESUMO
Wheat germ phosphoglycerate mutase, exposed to 3.4 M guanidinium chloride at 22 degrees C and pH 7.8, slowly undergoes time-dependent inactivation which can be fully reversed by adding excess Co2+ or Mn2+ to a 50-fold dilution of the denaturing medium. Titration of the denatured enzyme with either Co2+ or Mn2+ shows that wheat germ mutase preferentially binds Co2+. Assuming 1:1 complexation between metal atom and protein, the apparent dissociation constants (Kd) for E Co2+ and E Mn2+ at 22 degrees C and pH 8.7 are approximately 1.06 and 1.84, respectively. Other metal atoms (e.g., Cr2+, Cu2+, Fe2+, Fe3+, Mg2+, and Ni2+) have no effect in restoring the apoenzyme's catalytic activity. At low concentrations (0.11-0.23 mM) Zn2+ partially restores activity, but promotes protein precipitation at elevated concentrations. Evidence suggests that all bisphosphoglycerate-independent phosphoglycerate mutases require either an intra- or an extramolecular metal atom in order to function. Attempts to characterize wheat germ mutase as a glycoprotein have yielded negative results.
Assuntos
Bisfosfoglicerato Mutase/análise , Fosfotransferases/análise , Triticum/enzimologia , Cobalto/farmacologia , Guanidina , Guanidinas , Cinética , Manganês/farmacologia , Fatores de TempoRESUMO
A method is presented by which rat facial processes from different stages were obtained in pure fraction. The morphology, and protein and DNA contents in free dissected facial processes were determined. Facial processes of embryonic rats aged 9-15 days were analyzed by isoelectric focusing for their isoenzymic distribution of four enzymes: lactate dehydrogenase, creatine phosphokinase, fructose diphosphate aldolase and phosphoglycerate mutase. A dominance of LDH-5, LDH-4 and LDH-3 isoenzymes was observed. As a comparison, LDH isoenzymes from mandibular and maxillary processes of rat embryos aged 9-11 days only revealed LDH-5 and to a smaller extent LDH-4. The results support the presence of a prominent anaerobic metabolism in these tissues during early facial development. The change to LDH-3 development correlates well with the formation of new blood vessels. From the ninth embryonic day, isoenzyme BB of creatine phosphokinase was present and during days 10-15 MB and MM developed. Isoenzyme A4 of fructose diphosphate aldolase was present from the ninth embryonic day and isoenzymes A3C and A2C2 developed during days 10-15. From the tenth embryonic day, isoenzyme BB of phosphoglycerate mutase was present and during days 10-15 isoenzyme MB and MM developed. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal and medial nasal processes, and it preceded morphologic evidence of skeletal muscle formation.
Assuntos
Ossos Faciais/embriologia , Isoenzimas/análise , Animais , Bisfosfoglicerato Mutase/análise , Creatina Quinase/análise , Ossos Faciais/enzimologia , Frutose-Bifosfato Aldolase/análise , Camadas Germinativas/enzimologia , Focalização Isoelétrica , L-Lactato Desidrogenase/análise , Mandíbula/embriologia , Mandíbula/enzimologia , Maxila/embriologia , Maxila/enzimologia , Osso Nasal/embriologia , Osso Nasal/enzimologia , Ratos , Ratos EndogâmicosRESUMO
Purified phosphoglycerate mutase from pig skeletal muscle and 2,3-bisphosphoglycerate synthase-phosphatase from pig erythrocytes were hybridized "in vitro". The hybrid showed a behaviour on electrophoresis and on ion-exchange chromatography similar to that of a naturally occurring enzyme with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities present in pig skeletal and heart muscle. Both the hybrid and the muscle enzyme possess similar activities ratio. From these and previous data it is suggested that the six enzymatic forms with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities detected in mammalian tissues (Carreras et al. 1981, Comp. Biochem. Physiol. 70B, 477-485) result from combination of three subunits (types M, B and E).
Assuntos
Bisfosfoglicerato Mutase/análise , Isoenzimas/análise , Monoéster Fosfórico Hidrolases/análise , Fosfotransferases/análise , Animais , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Eritrócitos/enzimologia , Congelamento , Substâncias Macromoleculares , Músculos/enzimologia , Suínos , UltrafiltraçãoRESUMO
Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also presented which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.