RESUMO
A comparison of two methods of measuring liver mitochondrial redox state demonstrated that a linear correlation exists between acetoacetate/beta-hydroxybutyrate ratio in arterial blood (arterial ketone body ratio; AKBR) and oxidized flavoprotein/reduced pyridine nucleotide in human liver tissue (FP/PN) as measured by tissue fluorescence spectroscopy, such that [FP/PN] = 0.64 + 0.49 x [AKBR] (r = 0.84, P less than 0.001). This result supports the validity of AKBR as a method of measuring the hepatic mitochondrial redox state of pyridine nucleotide using arterial blood.
Assuntos
Acetoacetatos/sangue , Butiratos/sangue , Hidroxibutiratos/sangue , Mitocôndrias Hepáticas/metabolismo , Ácido 3-Hidroxibutírico , Flavoproteínas/sangue , Fluorescência , Congelamento , Humanos , NAD/sangue , Oxirredução , Sensibilidade e EspecificidadeRESUMO
A sensitive and specific chemiluminescence (CL) method with bacterial luciferase was adapted for accurate measurement of the flavins FAD and FMN in the membrane and cytosolic fractions of neutrophils prepared from pig and human blood. The FAD and FMN contents (FAD/FMN = 100:2) in the membranes were essentially the same in resting (R) and myristate-stimulated (S) cells, although O2(-)-generation was markedly enhanced exclusively in S membranes. The O2(-)-forming activity of S samples remained unchanged or even increased after washing the membranes with buffer, although one-third of the FAD was lost during washing (a decrease from 140 to 95 pmol/10(8) cell-equivalent (CE) during washing). The cytosol is known to contain at least three components that are essential for O2- production (p47-phox, p67-phox, and a G-protein), and that are translocated to membranes upon activation, but its flavin content was one tenth of that of the membranes. The cytosol was treated with fatty acids in the absence of membranes to induce substantial precipitation of p47-phox, p67-phox and a protein of 32 kDa. No difference relative to a solvent-control was noted in the low flavin content of the precipitate indicating that these cytosolic components are not flavoproteins. These results do not support the possibility of translocation of a cytosolic flavoprotein to the membrane upon activation of the respiratory burst.
Assuntos
Mononucleotídeo de Flavina/sangue , Flavina-Adenina Dinucleotídeo/sangue , Flavoproteínas/sangue , Medições Luminescentes , Neutrófilos/química , Animais , Membrana Celular/metabolismo , Citosol/metabolismo , Ácidos Graxos/farmacologia , Humanos , Luciferases/metabolismo , Ácido Mirístico , Ácidos Mirísticos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Superóxidos/sangue , SuínosRESUMO
It has been observed that the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin in murine host, designated as Dalton's lymphoma (DL), induces inhibition of various immune responses and is associated with an involution of thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes caused by induction of apoptosis with a decrease of CD4+CD8+, CD4+CD8- and CD4-CD8+ thymocytes. Here, we report that serum of DL-bearing mice contains soluble factors capable of inducing thymocyte apoptosis, the effectiveness of which increases with the progression of tumor growth. A decline of essential cytokines and hormones in the body due to their depletion by DL cells, which being a T-cell phenotype may have similar growth factor requirements, is ruled out by our results, suggesting additional apoptosis-inducing factors to be present in the tumor serum. Partial characterization of the serum to identify the biochemical nature of the putative serum-borne apoptosis inducing factor(s) showed that the same was proteinaceous. Further analysis of the sera of normal and DL-bearing mice by gel filtration using fast protein liquid chromatography (FPLC) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that protein profile in the two sera differed quantitatively as well as qualitatively. FPLC analysis could resolve six peaks in both the sera, out of which the peak containing protein(s) in the range of MW 35 kD showed a higher magnitude and apoptotic activity followed by peaks containing proteins of MW in the range of 67 and 116 kD respectively as compared to that of the corresponding peaks in the normal serum. These observations were also confirmed by SDS-PAGE, with the resolution of additional proteins in the range of 25-26 kD which were found to be absent in normal serum. Further, the paper discusses different possible factors that could be associated with the progression of DL growth.
Assuntos
Apoptose/efeitos dos fármacos , Flavoproteínas/sangue , Linfoma de Células T/sangue , Linfoma de Células T/patologia , Proteínas de Membrana/sangue , Timo/patologia , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose , Fatores Biológicos/sangue , Fatores Biológicos/farmacologia , Fatores Biológicos/fisiologia , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida , Feminino , Flavoproteínas/análise , Flavoproteínas/farmacologia , Linfoma de Células T/imunologia , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Dodecilsulfato de Sódio , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
The three-dimensional structure of the dimeric flavoenzyme glutathione reductase from human erythrocytes has been elucidated by an X-ray diffraction analysis at 0.3 nm resolution. The polypeptide chain has been traced, and the binding positions of FAD, NADP and glutathione have been determined. A mechanism for the electron transfer is discussed.
Assuntos
Glutationa Redutase , Sítios de Ligação , Catálise , Eritrócitos/enzimologia , Flavoproteínas/sangue , Glutationa Redutase/sangue , Humanos , Modelos Moleculares , NADP/metabolismo , Conformação Proteica , Difração de Raios XRESUMO
Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.
Assuntos
Flavoproteínas/sangue , NADH NADPH Oxirredutases/sangue , Neutrófilos/metabolismo , Animais , Membrana Celular/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Radicais Livres , Concentração de Íons de Hidrogênio , NADP/farmacologia , NADPH Oxidases , Oxirredução , Potenciometria , Suínos , TermodinâmicaRESUMO
The resolved flavoprotein and cytochrome b559 components of the NADPH dependent O2- . generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b559, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent O2- . generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b559, depleted of flavoprotein, demonstrated no measureable NADPH dependent O2- . generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b559 was rapidly oxidized by oxygen, as was the cytochrome b559 in the intact oxidase.
Assuntos
Grupo dos Citocromos b/sangue , Flavoproteínas/sangue , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/enzimologia , Aerobiose , Anaerobiose , Grupo dos Citocromos b/isolamento & purificação , Flavoproteínas/isolamento & purificação , Humanos , Cinética , NADH NADPH Oxirredutases/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.
Assuntos
Flavoproteínas/sangue , Doença Granulomatosa Crônica/sangue , NADP/farmacologia , Neutrófilos/enzimologia , Oxirredutases/sangue , Grupo dos Citocromos b/sangue , Flavina-Adenina Dinucleotídeo/sangue , Humanos , Masculino , Espectrometria de Fluorescência , Superóxidos/sangueRESUMO
Two cytosolic proteins, p47-phox and p67-phox, have been shown to be essential components of the NADPH-dependent oxidase of human neutrophils, although the specific role of each of these proteins in the multicomponent electron transport complex is undetermined. The superoxide-generating activity of this oxidase can be reproduced in a cell-free system, combining cytosol and membranes from unstimulated neutrophils in the presence of fatty acid and NADPH. In the present studies, cytosol was treated with myristic acid, arachidonic acid, or sodium dodecyl sulfate in the absence of membranes and the resultant precipitate collected by centrifugation and analyzed. Both p47-phox and p67-phox precipitated in the presence of fatty acid. However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated. These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH.
Assuntos
NADH NADPH Oxirredutases/sangue , Neutrófilos/enzimologia , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Membrana Celular/enzimologia , Citosol/enzimologia , Flavinas/sangue , Flavoproteínas/sangue , Humanos , Cinética , Ácido Mirístico , Ácidos Mirísticos/farmacologia , NADH NADPH Oxirredutases/isolamento & purificação , NADPH Oxidases , Dodecilsulfato de Sódio/farmacologiaRESUMO
Twenty-five patients suffering from chronic granulomatous disease (CGD) and their families were investigated. Defects in the superoxide generating system were characterized at the level of the heme-containing cytochrome b and of the FAD-containing flavoprotein, both localized in the plasma membrane of granulocytes. It was confirmed that in most of the typical cases (18 of 22), the complete inability of superoxide generation was associated with the absence of detectable cytochrome b. Mothers but not fathers of such male patients were characterized by a diminished content of cytochrome b, confirming that the affected gene is localized on the X chromosome. In contrast, the granulocytes of four other typical patients (two female and two male) contained normal amounts of cytochrome b, whereas oxidative activity was absent. Since no abnormality of oxidative activity as well as of cytochrome b was found in granulocytes of the mothers and fathers of these patients, an autosomal recessive mode of inheritance of the disease is probable. The flavoprotein deficiency found in the granulocytes of four male patients was always associated with an absence of detectable cytochrome b. This could indicate a structural relationship between flavoprotein and cytochrome b (e.g., a flavocytochrome). Three further patients with mild X-linked CGD contrasted with the patients with severe or classic X-linked disease; the oxidative activity of their phagocytes was diminished but not absent, and the cytochrome b present, albeit in small amounts.
Assuntos
Grupo dos Citocromos b/sangue , Flavoproteínas/sangue , Doença Granulomatosa Crônica/classificação , Adolescente , Pré-Escolar , Grupo dos Citocromos b/deficiência , Feminino , Flavina-Adenina Dinucleotídeo/sangue , Flavoproteínas/deficiência , Genes Recessivos , Ligação Genética , Granulócitos/metabolismo , Doença Granulomatosa Crônica/sangue , Doença Granulomatosa Crônica/genética , Humanos , Técnicas In Vitro , Lactente , Recém-Nascido , Masculino , Nitroazul de Tetrazólio/metabolismo , Superóxidos/sangue , Cromossomo XRESUMO
Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity were measured in G6PD deficiency, heterozygous beta-thalassaemia and controls. It is confirmed that, in contrast to the high stimulation of GR by FAD commonly found in in thalassaemia indicating red-cell deficiency of FAD, and shown here to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD deficiency, leading to high in vitro activity. Unexpectedly, on the other hand, low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN, confirmed by response to oral riboflavin, was found in the majority of subjects with G6PD deficiency, similar to that found in heterozygous beta-thalassaemia. Whereas this is explained in thalassaemia by an inherited slow red-cell metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and high FAD. When G6PD deficiency occurs with heterozygous beta-thalassaemia, GR is usually saturated with FAD as in G6PD deficiency alone, unless there is an inherited, very slow red-cell metabolism of riboflavin to FMN. The part played by GR in haemolytic crises in G6PD deficiency is discussed.
Assuntos
Flavoproteínas/sangue , Deficiência de Glucosefosfato Desidrogenase/enzimologia , Glutationa Redutase/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fosfato de Piridoxal/análogos & derivados , Piridoxaminafosfato Oxidase/metabolismo , Eritrócitos/enzimologia , Mononucleotídeo de Flavina/sangue , Flavina-Adenina Dinucleotídeo/sangue , Deficiência de Glucosefosfato Desidrogenase/tratamento farmacológico , Heterozigoto , Humanos , Linhagem , Fosfato de Piridoxal/sangue , Riboflavina/uso terapêutico , Talassemia/enzimologiaRESUMO
Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.
Assuntos
Grupo dos Citocromos b/sangue , Flavoproteínas/sangue , Neutrófilos/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/enzimologia , Membrana Celular/enzimologia , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Citosol/enzimologia , Flavoproteínas/química , Flavoproteínas/genética , Fungos/enzimologia , Dados de Sequência Molecular , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/enzimologia , Homologia de Sequência do Ácido Nucleico , Superóxidos/sangue , SuínosRESUMO
It has been observed that the progressive ascitic growth of a transplantable T cell lymphoma of spontaneous origin, designated as Dalton's lymphoma (DL), induces inhibition of various immune responses and is associated with an involution of the thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes, with a decrease in CD4+CD8+, CD4+CD8- and CD4-CD8+ thymocytes. Morphological evaluation of thymocytes from DL-bearing mice revealed that with the progression of DL, a majority of thymocytes exhibited morphological features characteristic of apoptotic cell death, which included contracted cell bodies, condensed, uniformly circumscribed and densely stained chromatin, and membrane-bound apoptotic bodies containing one or more nuclear fragments. Quantitative and qualitative analysis of the DNA extracted from the thymocytes of DL-bearing mice revealed DNA fragmentation that increased concomitantly with the progression of DL and showed an oligonucleosomal DNA ladder pattern upon agarose gel electrophoresis, a hallmark of apoptotic cell death. Attempts to identify apoptotic factor(s) showed that the serum of DL-bearing mice contained certain soluble factor(s) that augmented the induction of apoptotis in thymocytes in a time- and dose-dependent manner. Although DL cells or their products, such as DL-cell-conditioned medium or DL-cell-free ascitic fluid, could also induce apoptosis of thymocytes in vitro, the magnitude of the same was consistently lower than that induced by the serum of DL-bearing mice. Further, elucidation of the mechanism of apoptosis induction in thymocytes with respect to the involvement of apoptosis-related genes revealed that the death pathway followed an interleukin-1 beta-converting-enzyme-dependent, Fas-mediated apoptotic cascade, with a concomitant increase in the protein products of the bax, bad, p53, fas and fasL genes and cleavage of the 23-kD N-terminal fragment of Bcl-2 that exhibited Bax-like death effector properties.