RESUMO
BACKGROUND: Acantholysis in pemphigus vulgaris (PV) may be triggered by desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient results in distinct intracellular signalling patterns. OBJECTIVES: Based on our previous findings, we aimed to elucidate whether PV acantholysis in a mouse model may be mediated by activation of a disintegrin and metalloproteinase 10 (ADAM10). METHODS: We used three PV-IgG fractions from different patients containing high or low levels of anti-Dsg1 and anti-Dsg3 antibodies, and the presence or not of anti-desmocollin (Dsc) antibodies, using a passive transfer mouse model of PV. RESULTS: Although all of the PV-IgG fractions produced suprabasal acantholysis, only those containing anti-Dsg1/3, but not anti-Dsc2/3 antibodies, induced ADAM10 activation in a Src-dependent way, and an increase in the epidermal growth factor (EGF) receptor ligands EGF and betacellulin (BTC). In contrast, the presence of anti-Dsc2/3 antibodies, in addition to anti-Dsg1/3, triggered earlier and ADAM10-independent epidermal detachment, with no increase in EGF and BTC, which was associated with an earlier and more intense acantholysis. CONCLUSIONS: All PV-IgG fractions produced suprabasal acantholysis, but our results reveal that depending on the levels of anti-Dsg antibodies or the presence of non-Dsg antibodies, such as anti-Dsc, more severe cell-cell epidermal detachment will occur at different times, and in an ADAM10-dependent manner or not. Acantholysis in these different groups of patients with PV may be a consequence of the activation of specific intracellular mechanisms downstream of Autoantibodies binding to Dsg or non-Dsg proteins, and therefore more specific therapeutic approaches in PV should be used. What's already known about this topic? Suprabasal acantholysis in pemphigus vulgaris (PV) may be triggered by both desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient is associated with a distinct intracellular signalling pattern. What does this study add? In patients with PV with anti-Dsg3 and anti-Dsg1, but not anti-desmocollin (Dsc)3 antibodies, ADAM10 activation is induced in an Src-dependent way, together with an increase in the epidermal growth factor receptor (EGFR) ligands EGF and betacellulin. The presence of anti-Dsc3 antibodies triggers an earlier and ADAM10-independent acantholysis, without increasing EGFR ligands, and is associated with more severe epidermal detachment. Lower levels of anti-Dsc3 antibodies are associated with less severe acantholysis. What is the translational message? In some patients with PV, the severity and the timing for cell-cell detachment seem to depend on the level of anti-Dsg1/3 antibodies, although other as yet uncharacterized antibodies may also participate. These patients with PV would exhibit inhibition of acantholysis by Src, ADAM10, EGF and EGFR inhibitors. In other patients, the presence of non-Dsg antibodies, such as anti-Dsc2/3, would produce an earlier and more severe ADAM10-independent suprabasal acantholysis.
Assuntos
Acantólise , Autoanticorpos , Pênfigo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Animais , Desmogleína 1 , Desmogleína 3 , Humanos , Proteínas de Membrana , CamundongosRESUMO
BACKGROUND: Pemphigus vulgaris (PV) is a blistering autoimmune disease characterized by IgG autoantibodies against desmoglein 3. Nitric oxide synthases (NOS) may contribute to the increase of inflammation in tissues by the generation of nitrotyrosine residues (NTR). OBJECTIVES: To investigate whether the production of NTR mediated by NOS may participate in the development of inflammation and acantholysis in PV. METHODS: Mice were pretreated or not with NOS, tyrosine-kinase (TK) or nuclear factor (NF)-kappaB inhibitors, and then injected with PV-IgG. PV manifestations were examined in all mice. The expression of NTR, constitutive NOS (cNOS) [endothelial NOS (eNOS) and neuronal NOS (nNOS)], inducible NOS (iNOS) and NF-kappaB factor were studied in epidermis of mice using immunohistochemical techniques. RESULTS: After PV-IgG injection, expressions of NTR, iNOS, eNOS and nNOS increased in acantholytic cells, as did nuclear translocation of NF-kappaB in the basal cells of the epidermis. Pretreatment of mice with inhibitors of TK, nNOS and nonselective NOS, completely prevented NTR expression and the clinical and histological findings of PV in mice. TK inhibitor genistein inhibited both nNOS and iNOS expression on the membrane of basal keratinocytes, and nuclear translocation of NF-kappaB. CONCLUSIONS: Upregulation of cNOS and iNOS, NTR generation and nuclear translocation of NF-kappaB may contribute to increased inflammation and tissue damage in PV lesions. The absence of the clinical and histological findings of PV and NTR expression in mice injected with PV-IgG, through pretreatment with TK and nNOS inhibitors, provides compelling evidence that these signalling molecules should be considered as potential therapeutic targets in PV.
Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Pênfigo/enzimologia , Animais , Animais Recém-Nascidos , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , NF-kappa B/metabolismo , Regulação para CimaRESUMO
We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.
Assuntos
Autoantígenos/química , Doenças Autoimunes/imunologia , Monócitos/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Citocinas/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/química , Humanos , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-2/metabolismo , Dados de Sequência Molecular , Peptídeos/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
The presence of a DNase activity associated with secretion granules was detected in T4 and T8 lymphocytes from patients with autoimmune diseases. This activity was much higher in primary biliary cirrhosis (PBC) than in Graves' disease (GD) and multiple sclerosis (MS) or in healthy subjects. This granule associated DNase activity was Ca(2+)-dependent, inhibited by Zn2+, and higher at low pH; its molecular weight corresponded to 66kDa; it was more active with double-strand than single-strand DNA. Judging from its properties this enzyme differed from the three types of endonucleases described as involved in DNA fragmentation (DNase I, DNase II and NUC18). Flow cytometry analysis of T lymphocytes showed that DNase activity associated with CD4+ lymphocyte granules correlated with the ratio CD4+CD45RO+/CD4+CD45RA+ (memory and cytotoxic cells/naive cells, inducers of suppression). In contrast, T8 lymphocyte DNase activity correlated with the proportion of CD4+ lymphocytes with CD4+CD45RA- phenotype (helpers and inducers of cytotoxicity). The possible role of this DNase activity in the mechanisms of lysis or apoptosis mediated by CTL is discussed. We suggest that this DNase activity could be implicated in some of the alterations of the autoimmune response depending on cytotoxic T lymphocytes or T cell inducers of apoptosis.
Assuntos
Doenças Autoimunes/enzimologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Grânulos Citoplasmáticos/enzimologia , Desoxirribonucleases/metabolismo , Adulto , Apoptose/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD8-Positivos/ultraestrutura , Dano ao DNA , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Peptides from 10 to 22 amino acids containing sequences encompassed by Staphylococcus aureus protein A were synthesized. Some of these peptides, when present in cultures of lymphomononuclear cells from healthy donors or from cancer patients (melanoma, breast carcinoma, non-Hodgkin lymphoma and renal cell carcinoma) promoted: (i) changes in the phenotype of the lymphomononuclear population, (ii) stimulation of monocytes (release of IL-1 and TNF-alpha), and (iii) an increase in cytotoxicity against K562, Daudi and HT-29 cells. Isolated monocytes responded also to those peptides with a release of IL-1 and TNF alpha and an increase of cytotoxicity against HT-29 cells. It was found that the active peptides had the following structural pattern: a length of at least 15 amino-acid residues with a proline at position 6, valine, leucine, isoleucine, glycine, alanine or lysine at position 2, and glutamic or aspartic acid at position 11. Replacement of Pro at position 6 with any other residue turned the peptide inactive. Replacement of residues at positions 2 and 11 with amino-acid residues other than those required for activity resulted in compounds with a marked decrease in the immunomodulating properties described, or lacking these properties altogether.
Assuntos
Citotoxicidade Imunológica , Leucócitos Mononucleares/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Proteína Estafilocócica A/farmacologia , Sequência de Aminoácidos , Antígenos de Superfície/análise , Morte Celular , Células Cultivadas/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Interleucina-1/metabolismo , Dados de Sequência Molecular , Neoplasias/sangue , Peptídeos/química , Proteína Estafilocócica A/química , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A series of peptides of 15 amino acids with sequences contained in human extracellular matrix (ECM) proteins (fibronectin, laminin A, laminin B1, tenascin, undulin, alpha 1-chain of type IV and VIII collagen and alpha 2-chain of type VIII collagen) have been synthesized. The selected structures conformed to the following pattern: (i) Pro at position 6, (ii) Leu, Lys, Ile, Val, Ala or Gly at position 2, (iii) Glu or Asp at position 11. Fibronectin and the indicated peptides, when present in cultures of lymphomononuclear cells from healthy donors, promoted stimulation of monocytes manifested by a release of IL-1 alpha, IL-beta, IL-6 and TNF alpha; an increase in the percentage of cells expressing CD14, CD16, CD11b and CD14/CD16; an increase in cytotoxicity against HT-29. Cytotoxicity against K562 and Daudi cells (targets of NK and LAK cells) was also observed together with an increase in the percentage of cells expressing CD56, CD56/CD16 (corresponding to NK cells), and CD56/CD8 (corresponding to NK-like lymphocytes), indicating a stimulation of lymphocytes. Activated monocytes and lymphocytes contained a large number of granules with DNAse activity. These results suggest that at least some of the immunological properties of ECM proteins could be accounted for by motifs fulfilling a characteristic sequence pattern shared by all of them.
Assuntos
Proteínas da Matriz Extracelular/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Sequência de Aminoácidos , Células Cultivadas , Citocinas/biossíntese , Citotoxicidade Imunológica/efeitos dos fármacos , Proteínas da Matriz Extracelular/síntese química , Humanos , Dados de Sequência MolecularRESUMO
Redox titration of F1-ATPase from rat liver mitochondria referred to the modification of the hydrolytic activity on Mg-ATP has resulted in a three-step pattern, with three distinct jumps of activity separated by clear plateaus. The measured potentials ranged from -400 mV to +400 mV and were obtained by the addition of dithionite and ferricyanide. Electron exchange was facilitated with a mixture of different redox mediators. At pH 7.4 the midpoint potentials were +210 mV, +40 mV and -230 mV. These three midpoint potentials were displaced towards more negative values by 2,4-dinitrophenol or by an increase of the pH of the medium. The titration curves were described by n = 2 Nernst equations.
Assuntos
Mitocôndrias Hepáticas/enzimologia , ATPases Translocadoras de Prótons/fisiologia , Animais , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Oxirredução , RatosRESUMO
Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein-3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell-cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase-3 activation, Bcl-2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes.
Assuntos
Apoptose/imunologia , Autoanticorpos/farmacologia , Queratinócitos/citologia , Queratinócitos/imunologia , Pênfigo/imunologia , Autoanticorpos/imunologia , Caspase 3 , Caspases/metabolismo , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fragmentação do DNA , Relação Dose-Resposta Imunológica , Imunoglobulina G/metabolismo , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Microscopia de Fluorescência , Testes de Precipitina , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Proteína X Associada a bcl-2RESUMO
Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15-mers containing a characteristic 2-6-11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.
Assuntos
Aminoácido Oxirredutases/biossíntese , Proteínas da Matriz Extracelular/fisiologia , Leucócitos Mononucleares/enzimologia , Sequência de Aminoácidos , Células Cultivadas , Indução Enzimática , Humanos , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/fisiologia , Proteína Estafilocócica A/farmacologiaRESUMO
The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.
Assuntos
Colágeno Tipo I/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepatócitos/citologia , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Piridinas/farmacologia , Ratos , Esfingomielina Fosfodiesterase/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Phenotypes of 38 samples of mononuclear (PBMC) cells from 11 different patients with pemphigus vulgaris (PV) at different stages of the disease were explored looking for a possible relationship between cell immunity, mucocutaneous or mucosal lesion intensity and capacity of serum autoantibodies to elicit the disease in mice. PBMC from 5 patients with mucocutaneous lesions and sera with IgG capable of inducing the disease in neonatal mice had a high proportion of mature monocytes with CD14low DRhigh, and co-expressing CD16 and CD11b. In addition, a high proportion of CD19+CD5+ activated B cells and a very low proportion of naive CD4+CD45RA+ and CD8+CD11b+ T lymphocytes was observed. Monocytes from these patients expressed inducible nitric oxide synthase (iNOS). In contrast, PBMC from 6 patients, with lesions restricted to mucosal membranes and IgG lacking the capacity to induce the disease in mice, contained a high proportion of CD14high DRlow co-expressing CD16 circulating macrophages, CD8+CD11b+ T cells, and a low proportion of activated B lymphocytes. The results suggest a possible association between proportion of different antigen presenting cells (monocytes with high HLA-DR and low CD14 expression and activated B lymphocytes, or differentiated monocytes/macrophages), type of PV and capacity of serum autoantibodies to elicit the disease in mice.
Assuntos
Leucócitos Mononucleares/imunologia , Pênfigo/imunologia , Acantólise/etiologia , Acantólise/imunologia , Acantólise/patologia , Animais , Animais Recém-Nascidos , Autoanticorpos/administração & dosagem , Autoanticorpos/sangue , Modelos Animais de Doenças , Antígenos HLA-DR/sangue , Humanos , Imunização Passiva , Imunoglobulina G/administração & dosagem , Imunoglobulina G/sangue , Imunofenotipagem , Leucócitos Mononucleares/enzimologia , Receptores de Lipopolissacarídeos/sangue , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/classificação , Monócitos/enzimologia , Monócitos/imunologia , Óxido Nítrico Sintase/sangue , Óxido Nítrico Sintase Tipo II , Pênfigo/enzimologia , Pênfigo/patologiaRESUMO
The Ca²( +) -dependent ATPase activity of spinach chloroplast coupling factor 1 (CF1) is activated by treatment with dithiothreitol (DDT). If excess of this reagent is eliminated by gel filtration, an Eadie-Hofstee biphasic plot is obtained. These results are consistent with the existence of two active forms of the enzyme governed by the redox state. We have observed that SDS-polyacrylamide gel electrophoresis pattern is affected by the pretreatment of the samples under those two different conditions. Spontaneous activation of the samples, due to a limited proteolytic process, has also been detected. In this case the electrophoretic pattern was also affected. The protease implied in this process could be a cystein protease co-isolated with CF1. These observations suggest that limited proteolysis, as well as redox-induced changes, are involved in the physiological regulation of the enzyme.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Spinacia oleracea/enzimologia , Ditiotreitol/química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Oxirredução , Proteínas/metabolismoRESUMO
We analyzed the phenotypic characteristics of PBMC from 34 patients with Graves' disease (GD) at different stages of the disease to explore the sequence of immunological events associated with it. In all cases their monocytes were in a state of activation and differentiation more advanced than those of a group of 23 healthy individuals. Strikingly, some patients had CD14++DR- immature monocytes, which were absent in healthy individuals. CD14+CD16+DRhigh monocytes were more abundant in patients. We found a positive correlation between the CD14++DR- monocyte and CD4+CD45RA- helper cells and a negative correlation between the same monocyte subset and CD4+CD45RA+ naive cells. CD14+/++DRlow monocytes directly correlated with this latter T4 subset and CD14+ CD16+DRhigh with CD4+CD45RO+ memory lymphocytes. There was also a positive correlation between memory T4 cells and the subset of activated B lymphocytes (CD19+CD5+) and suppressor T8 cells (CD8+CD11b+). T8 cytotoxic cells (CD8+CD11b-) positively correlated with T4 naive cells. The circulating levels of T3 and TSI (thyroid-stimulating immunoglobulin) directly correlated with a decrease in naive cells and an increase in T8 suppressors. The results suggest that the imbalance suppression/cytotoxicity in GD may be due to a reiterated presentation of autoantigens, or mimetic antigens, to T helpers by mature monocytes and activated B cells.
Assuntos
Linfócitos B/imunologia , Doença de Graves/imunologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Antígenos CD/análise , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Seguimentos , Humanos , Interleucina-2/imunologia , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Fenótipo , Glândula Tireoide/imunologiaRESUMO
Synthetic peptides with sequences present in extracellular matrix protein fibronectin have been described to stimulate human monocytes. We describe now that one of these peptides, FN6, induces apoptotic effects on monocytes and we investigate the molecular mechanisms involved in the regulation of this response. Incubation of monocytes with FN6 induces the activation of the small GTPase Rac. In turn, Rac mediates the increase of both JNK and p38 activities in a sustained fashion, as well as the phosphorylation levels of their respective substrates c-Jun and ATF-2. FN6 also stimulates caspases -9 and -3 and the delayed proteolysis of its substrates PARP and D4-GDI. In addition, initiator caspases-1 and -5 were activated by FN6 treatment of monocytes but, in contrast to that observed for caspases-9 and -3, this effect was not dependent on JNK or p38 activities. These kinases also mediated the increase of Bax levels, but only in some conditions Bcl-2 depletion caused by the peptide. Moreover, whereas initially only caspase-1 is involved in caspase-3 activation, later on caspase-9 seems also to participate. Therefore, we demonstrate that FN6 stimulation allows multiple, JNK and p38-dependent and -independent interacting signals to regulate the apoptotic response in human monocytes.
Assuntos
Apoptose/efeitos dos fármacos , Fibronectinas/química , Monócitos/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/citologia , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.
Assuntos
ATPases Transportadoras de Cálcio/química , Cloroplastos/enzimologia , Proteínas de Plantas/química , ATPases Translocadoras de Prótons/química , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Sítio Alostérico , Ânions/farmacologia , Sítios de Ligação , ATPases Transportadoras de Cálcio/metabolismo , Catálise , Ativação Enzimática/efeitos dos fármacos , Fluoresceína-5-Isotiocianato/farmacologia , Cinética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tripsina/farmacologiaRESUMO
Peroxidation induced by ascorbate on phospholipids of isolated rat liver microsomes were accompanied by losses in glucose-6-phosphatase activity (EC 3.1.3.9.). The existence of marked differences in the degradation rate for each phospholipid suggests a relationship between the alteration of phosphatidylcholine containing one saturated and one unsaturated fatty acid and the decrease in activity of glucose-6-phosphatase; the inactivation of this enzyme was unrelated to the alteration of other phospholipids. These results support the idea that glucose-6-phosphatase and molecules of phosphatidylcholine having one saturated and one unsaturated fatty acid are in close apposition within the microsomal membrane.
Assuntos
Ácido Ascórbico/metabolismo , Glucose-6-Fosfatase/antagonistas & inibidores , Membranas/metabolismo , Microssomos Hepáticos/enzimologia , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Animais , Masculino , Membranas/enzimologia , RatosRESUMO
The effect of activating anions on the hydrolysis of ATP catalyzed by mitochondrial ATPase was higher on the oxidized than on the reduced form of the enzyme. On the contrary the effect of inhibitory anions on this reaction was more manifest on the reduced form of the enzyme. Kinetic data show that both activating and inhibitory anions compete for the same sites of the ATPase. A unifying mechanism of action is suggested according to which the anions could establish coordination bonds with the suggested iron atoms of the catalytic site. The preferential displacement of electrons of such bonds towards the ligand, or towards the metal atom, would lead respectively to an inhibition or to an activation of the enzyme.
Assuntos
Adenosina Trifosfatases/metabolismo , Ânions/farmacologia , Bicarbonatos/farmacologia , Mitocôndrias Hepáticas/enzimologia , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Sítios de Ligação , Ligação Competitiva , Ativação Enzimática , Cinética , Ligantes , Oxirredução , ATPases Translocadoras de Prótons , RatosRESUMO
The extent of stimulation of the hydrolytic activity of mitochondrial ATPase by the reducing agent dithionite has been found to depend on substrate concentration both for the membrane bound enzyme and for the isolated and purified F1ATPase. The results suggest the existence of three catalytic sites differing in their standard reduction potential. The activating effect of free ATP on the hydrolytic activity of rat liver F1-ATPase has been found to be more pronounced on the reduced form of the enzyme. On the contrary, the inhibitory effect of ADP was higher on the oxidized form of F1-ATPase. Citrate has also been found to be an inhibitor of F1-ATPase; its effect was more pronounced on the reduced form of the enzyme, and exhibited a competitive pattern of inhibition with respect to free ATP. The results obtained have been interpreted in the sense that free ATP and ADP may be modifying the standard reduction potential of the enzyme, and suggest the existence of three independent redox cycles in ATPase governed by the exchange of ADP and Pi for the newly synthesized ATP.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Ditionita/farmacologia , Mitocôndrias Hepáticas/enzimologia , Sulfitos/farmacologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Catálise , Citratos/farmacologia , Oxirredução , RatosRESUMO
A series of uncouplers and inhibitors of oxidative phosphorylation have been studied with regard to their effect on the hydrolytic activity of the reduced and oxidized forms of isolated or membrane-bound mitochondrial ATPase. Uncouplers (2,4-dinitrophenol, dicoumarol), which are also activators of the hydrolytic activity of ATPase, were more potent activators on the oxidized form of the enzyme. Inhibitors of oxidative phosphorylation (oligomycin, azide and amytal) had a more potent inhibitory effect on the hydrolytic activity of ATPase in its reduced form. Purified F1-ATPase, oligomycin insensitive in the oxidized form of the enzyme, became sensitive to oligomycin in the reduced form. An interpretation of the results suggests the presence of a mechanism that unifies the action of these different compounds on the synthesis and hydrolysis of ATP catalyzed by mitochondrial ATPase.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Desacopladores/farmacologia , Adenosina Trifosfatases/biossíntese , Animais , Oxirredução/efeitos dos fármacos , RatosRESUMO
1. Pyridoxal-5'-phosphate (PLP), a marker of primary amines, bound covalently to S-1 in an approximate ratio of 1:1. PLP was localized within the tryptic 25,000 mol. wt fragment. 2. Fluorescein isothiocyanate (FITC), which is known to bind covalently to primary amino groups located at nucleotide binding sites, readily bound to S-1 in a 2:1 ratio. FITC was localized within the 20,000 and 50,000 mol. wt tryptic peptides, in a ratio of 1:1 in each one. 3. These results are consistent with the existence of nucleotide binding sites on myosin different from those of the catalytic sites.