Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
G3 (Bethesda) ; 13(8)2023 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-37284815

RESUMO

Phase separation is a major mechanism of macromolecular condensation within cells. A frequently chosen tool for global disruption of phase separation via weak hydrophobic interactions is treatment with 1,6-hexanediol. This study evaluates the cytotoxic and genotoxic effects of treating live fission yeast with 1,6-hexanediol. We find that 1,6-hexanediol causes a drastic decrease in cell survival and growth rate. We also see a reduction in HP1 protein foci and increase in DNA damage foci. However, there is no evidence for increased genomic instability in two classically phase-separated domains, the heterochromatic pericentromere and the nucleolar rDNA repeats. This study reveals that 1,6-hexanediol is a blunt tool for phase separation inhibition and its secondary effects must be taken into consideration during its in vivo use.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Humanos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Heterocromatina/metabolismo , Instabilidade Genômica
2.
J Cell Sci ; 136(14)2023 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-37259831

RESUMO

During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.


Assuntos
Dictyostelium , Dictyostelium/metabolismo , Fosfatos/metabolismo , Fosfatos/farmacologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proliferação de Células , Testes Genéticos
3.
Biol Open ; 10(2)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33579693

RESUMO

Studies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Genoma Fúngico , Instabilidade Genômica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Reparo do DNA , Imunofluorescência , Ligação Proteica , Transporte Proteico , Imagem com Lapso de Tempo
4.
Environ Pollut ; 240: 875-883, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29787978

RESUMO

Intertidal creeks form the primary hydrologic link between estuaries and land-based activities on barrier islands. Fecal indicators Enterococcus spp. (Entero1), pathogens Shigella spp. (ipaH), Salmonella spp. (invA), E. coli of EHEC/EPEC groups (eaeA), E. coli of EAEC, EIEC, and UPEC groups (set1B), E. coli of STEC group (stx1); and tetracycline resistance genes (tet(B), tet(C), tet(D), tet(E), tet(K), tet(Q), tet(W), and tet(X); TRG) were detected in the headwater of Oakdale Creek (Sapelo Island, GA) receiving runoffs from Hog Hammock village. Excavation of drainage ditches around the village caused a high increase in the incidence of the above determinants. Water samples were collected from the headwater, transferred to diffusion chambers, submersed in the headwater, saltmarsh, and mouth of the creek; and the determinants were monitored for 3 winter months. With some exceptions, their persistence decreased in order headwater > saltmarsh > mouth. Genes associated with Enterococcus spp. were the most persistent at all the sites, following in the headwater with determinants for Salmonella spp. and E. coli of EAEC, EIEC, and UPEC groups. In the mouth, the most persistent gene was eaeA indicating EHEC, EPEC, and STEC. Tet(B) and tet(C) persisted the longest in headwater and saltmarsh. No TRG persisted after 11 days in the mouth. Most determinants revealed correlations with temperature and pH, and inverse correlations with dissolved oxygen. Decay rates of the above determinants varied in the range of -0.02 to -0.81/day, and were up to 40 folds higher in the saltmarsh and mouth than in the headwater. Our data demonstrated that water parameters could to some extent predict a general trend in the fate of virulence and antibiotic resistance determinants in tidal creek tributaries but strongly suggested that their persistence in these tributaries cannot be predicted from that of enterococci, or extrapolated from one biological contaminant to another.


Assuntos
Farmacorresistência Bacteriana/genética , Enterococcus/crescimento & desenvolvimento , Monitoramento Ambiental , Genes Bacterianos , Rios/microbiologia , Antibacterianos , Bactérias/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fezes/microbiologia , Testes de Sensibilidade Microbiana , Tetraciclina , Resistência a Tetraciclina/genética , Virulência
5.
Stud Health Technol Inform ; 181: 27-31, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22954822

RESUMO

We are entering a new age where people routinely visit, inhabit, play in and learn within virtual worlds (VWs). One in eight people worldwide are VW participants, according to the latest 2011 figures from KZERO [1]. VWs are also emerging as a new and advanced form of telehealth care delivery. In addition to existing telehealth care advantages; VWs feature three powerful affordances that can benefit a wide range of physical and psychological issues. First, the highly social nature of VWs encourages social networking and the formation of essential support groups. Secondly, the type of spaces that have been proven in the physical world to promote psychological health and well-being can be virtually recreated. Finally, research suggests that embodied avatar representation within VWs can affect users psychologically and physically. These three aspects of VWs can be leveraged for enhanced patient-client interactions, spaces that promote healing and positive responses, and avatar activities that transfer real benefits from the virtual to the physical world. This paper explains the mounting evidence behind these claims and provides examples of VWs as an innovative and compelling form of telehealth care destined to become commonplace in the future.


Assuntos
Simulação por Computador , Rede Social , Telemedicina/tendências , Interface Usuário-Computador , Humanos , Autoimagem , Apoio Social , Jogos de Vídeo
6.
NMR Biomed ; 13(6): 349-60, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11002314

RESUMO

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2). Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate. A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity.


Assuntos
Metabolismo Energético , Neoplasias Mamárias Experimentais/metabolismo , Animais , Ciclo do Ácido Cítrico , Feminino , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Espectroscopia de Ressonância Magnética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Propionatos/metabolismo , Esferoides Celulares , Células Tumorais Cultivadas
7.
Biochem J ; 350 Pt 2: 353-9, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10947948

RESUMO

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.


Assuntos
Fosfatidilinositol 3-Quinases/química , Androstadienos/farmacologia , Animais , Catálise , Domínio Catalítico , Bovinos , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Testes de Precipitina , Isoformas de Proteínas , Proteínas Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Transfecção , Wortmanina
8.
J Biol Chem ; 270(14): 7999-8008, 1995 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-7713900

RESUMO

Absolute metabolic fluxes in isolated perfused hearts have been determined by a nonlinear least squares analysis of glutamate labeling kinetics from [1-13C]glucose, [4-13C]beta-hydroxybutyrate, or [2-13C]acetate using 13C NMR spectroscopy. With glucose as substrate, the malate-aspartate shuttle flux was too slow to account for the reducing equivalents generated by glycolysis and to predict the observed oxygen consumption rate. For acetate and beta-hydroxybutyrate, the malate-aspartate shuttle had to be reversed for the network to agree with the observed oxygen consumption and glutamate labeling. Thus, an additional redox shuttle was required to reoxidize the NADH produced by cytoplasmic malate dehydrogenase. Using this model there was good agreement between the experimentally determined oxygen consumption and glutamate labeling and the calculated values of these parameters from the model for all substrates. The contribution of exogenous substrate to the overall tricarboxylic acid (TCA) cycle flux, 89.6 +/- 6.5% (mean +/- S.D.) as measured in the tissue extracts compared well with 91.4 +/- 4.2% calculated by the model. The ratio of TCA cycle flux to oxygen consumption for acetate, was 2.2 +/- 0.1, indicating that NADH production is principally accounted for by TCA cycle flux. For glucose or beta-hydroxybutyrate, this ratio was 2.9 +/- 0.2, consistent with the existence of other NADH producing reactions (e.g. glycolysis, beta-hydroxybutyrate oxidation).


Assuntos
Ácido Glutâmico/metabolismo , Miocárdio/metabolismo , Animais , Técnicas In Vitro , Cinética , Análise dos Mínimos Quadrados , Espectroscopia de Ressonância Magnética , Masculino , Oxigênio/metabolismo , Perfusão , Ratos , Ratos Sprague-Dawley
9.
Biochemistry ; 33(17): 5335-46, 1994 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-8172907

RESUMO

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand. Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
DNA/química , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Guanina/análogos & derivados , Conformação de Ácido Nucleico , Sequência de Bases , Citosina/metabolismo , DNA/genética , Primers do DNA/síntese química , Exodesoxirribonuclease V , Guanina/química , Guanina/metabolismo , Cinética , Matemática , Modelos Teóricos , Dados de Sequência Molecular , Moldes Genéticos , Timina/metabolismo
11.
Br J Pharmacol ; 86(1): 43-53, 1985 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-4052729

RESUMO

The cardiovascular effects of the opioid mixed agonist-antagonist, meptazinol, and the opioid antagonist, naloxone, have been evaluated in conscious rats, anaesthetized rats and anaesthetized cats following the induction of haemorrhagic shock. The mean arterial pressure of conscious rats decreased by 17-29 mmHg following a haemorrhage of 20% of blood volume. Meptazinol (17 mg kg-1, i.m.) administered after haemorrhage evoked a rapid and sustained increase in mean arterial pressure to pre-haemorrhage levels. Naloxone (10 mg kg-1, i.v.) also increased mean arterial pressure to a level significantly higher than post-haemorrhage values. Neither haemorrhage nor subsequent drug treatments evoked significant changes in the heart rates of conscious rats. In anaesthetized rats, 20% haemorrhage evoked decreases in mean arterial pressure, heart rate and cardiac output. Blood flow to the heart, skin, skeletal muscle, kidneys, spleen and liver (arterial) was decreased. Meptazinol and naloxone increased blood pressure and total peripheral resistance, but did not significantly alter heart rate or cardiac output. Hepatic arterial flow decreased further in both drug and vehicle treated groups. In addition meptazinol slightly reduced skeletal muscle flow. In anaesthetized cats 40% haemorrhage decreased mean arterial pressure by 46 +/- 3 mmHg. An intravenous infusion of either meptazinol or naloxone (cumulative 2 mg kg-1, i.v.) partially restored blood pressure. In experimental animal models of haemorrhagic shock, meptazinol has a similar cardiovascular profile to naloxone. The established analgesic activity of meptazinol may confer an advantage in some shock states.


Assuntos
Azepinas/farmacologia , Hemodinâmica/efeitos dos fármacos , Meptazinol/farmacologia , Naloxona/farmacologia , Choque Hemorrágico/fisiopatologia , Anestesia , Animais , Débito Cardíaco/efeitos dos fármacos , Gatos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Especificidade da Espécie , Fatores de Tempo , Resistência Vascular/efeitos dos fármacos
12.
J Biol Chem ; 258(22): 13785-94, 1983 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-6643454

RESUMO

Rat hearts have been perfused in vitro with 5 mM glucose and either 5 mM acetate or 1 mM pyruvate to achieve steady state conditions, followed by replacement of the acetate with 90% enriched [2-13C]acetate or pyruvate with 90% enriched [3-13C]pyruvate. The hearts were frozen different times after addition of 13C-substrate and neutralized perchloric acid extracts from three pooled hearts per time point were used to obtain high resolution proton-decoupled 13C NMR spectra at 90.55 MHz. The 13C fractional enrichment of individual carbons of different metabolites was calculated from the area of the resolved resonances after correction for nuclear Overhauser enhancement and saturation effects. A mathematical flux model of the citric acid cycle and ancillary transamination reactions was constructed with the FACSIMILE program, and used to solve unknown flux parameters with constant pool sizes by nonlinear least squares analysis of the approximately 200 simultaneous differential equations required to describe the reactions. With [2-13C] acetate as substrate, resonances and line splittings due to 13C-13C spin coupling of the C-2, C-3, and C-4 carbons of glutamate were well resolved. The half-times to reach maximum 13C enrichment were 2.6 min for glutamate C-4 and 8 min for glutamate C-2 and C-3. From these data, a well determined citric acid cycle flux of 8.3 mumol/g dry weight X min was calculated for an observed oxygen consumption of 31 mumol/g dry weight X min. With [3-13C]pyruvate as substrate, resonances of aspartate C-2 and C-3 and of alanine C-3 were well resolved in addition to those of glutamate C-2, C-3, and C-4. Nonlinear least squares fitting of these data to the model gave nonrandomly distributed residuals for the 13C fractional enrichments of glutamate C-4, suggesting an incomplete model, but a well determined cycle flux of 11.9 mumol/g dry weight X min for an oxygen uptake of 35 mumol/g dry weight X min. Our studies demonstrate the practicality of 13C NMR, used in conjunction with mathematical modeling, for the measurement of metabolic flux parameters in living systems.


Assuntos
Ciclo do Ácido Cítrico , Miocárdio/metabolismo , Animais , Isótopos de Carbono , Computadores , Marcação por Isótopo/métodos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Matemática , Modelos Biológicos , Perfusão , Ratos
13.
Biochem J ; 194(1): 215-28, 1981 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-6975619

RESUMO

1. The activity of creatine kinase in intact anaerobic frog muscle at 4 degrees C at rest and during contraction was investigated by using saturation-transfer 31P n.m.r. 2. At rest, the measured forward (phosphocreatine to ATP) reaction flux was 1.7 X 10(-3) M . s-1 and the backward flux was 1.2 X 10(-3) M . s-1. The large magnitude of both fluxes shows that creatine kinase is active in resting muscle, so the observed constancy of [phosphocreatine] demonstrates that the enzyme and its substrates are at equilibrium. 3. The apparent discrepancy between the fluxes must arise largely from an underestimation of the backward flux resulting from interaction of ATP with other systems, e.g. via adenylate kinase. For purposes of further calculation we have therefore adopted 1.6 X 10(-3) M . s-1 as an estimate of both fluxes. 4. During contraction, when the creatine kinase reaction is no longer at equilibrium, the net rate of phosphocreatine breakdown, estimated directly from the change in area of the inorganic phosphate peak, was 0.75 X 10(-3) M . s-1. Saturation transfer indicates that the forward reaction flux remains at approx. 1.6 X 10(-3) M . s-1 and the backward flux decreases to about 0.85 X 10(-3) M . s-1. 5. The activity of creatine kinase during contraction is large enough to account for the well-established observation that, during contraction, the concentration of ATP falls by less than 2-3%. The reaction catalysed by creatine kinase is driven forward during contraction by the large relative increase in the concentration of free ADP, which is more than doubled. 6. The observation that the forward flux does not increase during contraction and that the backward flux decreases can most simply be explained on the basis of competition of reactants for a limited amount of enzyme.


Assuntos
Creatina Quinase/metabolismo , Músculos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Cinética , Espectroscopia de Ressonância Magnética/métodos , Contração Muscular , Fosfocreatina/metabolismo , Rana temporaria
14.
Biochim Biophys Acta ; 590(1): 34-49, 1980 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-6243973

RESUMO

The kinetics and thermodynamics of the reaction of mixed valence state membrane-bound cytochrome oxidase with CO over the 178-203 K range has been studied by multichannel optical spectroscopy at three wavelength pairs (444-463 nm in the Soret region, and 590-630 and 608-630 nm in the alpha region) and analysed by non-linear optimization techniques. As in the case of the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore, G.M. and Chance, E.M. (1978) Biochem J. 175, 709-725), the normalized progress curves at the three wavelength pairs are significantly different indicating, on the basis of Beer's law, the presence of a minimum of three optically distinct species. The only model that satisfies the triple statistical requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters, is a two species sequential mechanism: flash photolysis of the mixed valence state cytochrome oxidase-CO complex (species IIMC) yields unliganded mixed valence state cytochrome oxidase (species EM) and free CO which then recombine to form species IMC; species IMC is then converted into species IIMC. All the thermodynamic parameters describing the model are calculated and compared to those obtained for the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore and Chance (1978) Biochem. J. 175, 709-725). Although there are some qualitative similarities in the kinetics and thermodynamics of the reactions of mixed valence state (alpha 23+Cu+B.ALPHA 3+Cu2+A) and fully reduced (a3 2+Cu B + . a2+Cu A+) cytochrome oxidase with CO, there are large and significant quantitative differences in zero-point activation energies and frequency factors; over the temperature range studied, the mixed valence state cytochrome oxidase-CO reaction is found to proceed at a significantly slower rate than the fully reduced cytochrome oxidase-CO reaction. These differences indicate that changing the valence states of cytochrome a and CuA has a significant effect on the CO binding properties of cytochrome a 3 and possibly CuB.


Assuntos
Monóxido de Carbono/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Cátions , Cobre/metabolismo , Cinética , Membranas/enzimologia , Oxirredução , Fotólise , Análise Espectral , Temperatura , Termodinâmica
16.
Biochem J ; 177(2): 613-21, 1979 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-219847

RESUMO

The kinetics of the reaction of fully reduced membrane-bound cytochrome oxidase with O2 obtained in the Soret, alpha and near-i.r. regions were analysed, and the contributions of the three intermediates of the reaction [Clore & Chance (1978) Biochem. J. 173, 799--810] to seven wavelength pairs (430--463, 444--463, 590--630, 608--630, 740--940, 790--940 and 830--940 nm) were determined. The nature of the intermediates is discussed on the basis of the data in the present paper together with data in the literature from optical wavelength scanning, e.p.r., i.r. and magnetic-susceptibility studies.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Oxigênio , Membrana Celular/enzimologia , Cinética , Modelos Químicos , Oxirredução , Espectrofotometria , Espectrofotometria Infravermelho , Temperatura
19.
Biochem J ; 175(2): 709-25, 1978 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-217348

RESUMO

1. The results of non-linear optimization studies on the mechanism of reaction of solid-state fully reduced membrane-bound cytochrome oxidase with CO over the 178--203 K range are presented. The analysis is carried out on data obtained by dual-wavelength multichannel spectroscopy at three wavelength pairs (444--463 nm, 590--630 nm and 608--630 nm), which yield three distinct progress curves. The only model that satisfies the triple requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters is a two-species sequential mechanism: flash photolysis yields unliganded cytochrome oxidase and free CO, which then recombine to form species Ic; Ic is then converted into species IIc, which is identical with the cytochrome oxidase-CO complex existing before flash photolysis. All the thermodynamic parameters describing this model are calculated. 2. On the basis of the data obtained from this paper, together with data from potentiometric studies, magnetic susceptibility measurements and i.r. spectroscopy, the chemical identity of the species is suggested.


Assuntos
Monóxido de Carbono/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Sítios de Ligação , Ativação Enzimática , Cinética , Modelos Químicos , Fotólise , Análise Espectral , Temperatura , Termodinâmica
20.
Biochem J ; 173(3): 799-810, 1978 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-213052

RESUMO

1. The results of non-linear optimization studies on the mechanism of reaction of fully reduced cytochrome oxidase with O2 at 176K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at three wavelength pairs (604-630, 608-630 and 830-940 nm) and at three O2 concentrations (60, 200 and 1180 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, good determination of the optimized parameters and a random distribution of residuals is a three-species sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from low-temperature trapping, e.p.r. and magnetic-susceptibility studies, the possible valence states of the metal centres in each of the intermediates are discussed.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Oxigênio , Fenômenos Químicos , Química , Temperatura Baixa , Cobre , Heme , Cinética , Modelos Químicos , Oxirredução , Análise Espectral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...