RESUMO
Bark beetles (Curculionidae: Scolytinae) and associated microorganisms must overcome a complex tree's defence system, which includes toxic monoterpenes, to successfully complete their life cycle. A number of studies have suggested these microorganisms could have ecological roles related with the nutrition, detoxification, and semiochemical production. In particular, in filamentous fungi symbionts, cytochrome P450 (CYP) have been involved with terpenoid detoxification and biotransformation processes. Candida oregonensis has been isolated from the gut, ovaries, and frass of different bark beetle species, and it is a dominant species in the Dendroctonus rhizophagus gut. In this study, we identify, characterise, and infer the phylogenetic relationships of C. oregonensis CYP genes. The results indicate that the cytochrome P450 complement (CYPome) is composed of nine genes (CYP51F1, CYP61A1, CYP56D1, CYP52A59, CYP52A60, CYP52A61, CYP52A62, CYP5217A8, and CYP5217B1), which might participate in primary metabolic reactions such as sterol biosynthesis, biodegradation of xenobiotic, and resistance to environmental stress. The prediction of the cellular location suggests that these CYPs to be anchored to the plasma membrane, membranes of the endoplasmic reticulum, mitochondria, and peroxisomes. These findings lay the foundation for future studies about the functional role of P450s, not only for yeasts, but also for the insects with which they interact.
Assuntos
Candida/classificação , Candida/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Filogenia , Gorgulhos/microbiologia , Animais , Candida/genética , Candida/isolamento & purificação , Membrana Celular/enzimologia , Trato Gastrointestinal/microbiologia , Membranas Intracelulares/enzimologia , Homologia de SequênciaRESUMO
In Trypanosoma cruzi three isoenzymes of phosphoglycerate kinase (PGK) are found which are simultaneously expressed: the cytosolic isoenzyme PGKB as well as two glycosomal enzymes, PGKA and PGKC. In this paper, we show that PGKA in T. cruzi epimastigotes is associated to the glycosomal membrane; it is responsible for about 23% of the glycosomal PGK activity, the fraction that remains in the pellet after osmotic shock treatment of purified organelles, in contrast to the 77% soluble activity that is mainly attributed to PGKC. Antibodies against the unique 80 amino-acid insertion of PGKA blocked almost completely the glucose consumption by epimastigotes that were partially permeabilized with digitonin. These results indicate that PGKA is the predominant isoenzyme for sustaining glycolysis through the glycosomes of these parasites.
Assuntos
Glucose/metabolismo , Membranas Intracelulares/enzimologia , Microcorpos/enzimologia , Fosfoglicerato Quinase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Transporte Biológico , Doença de Chagas/parasitologia , Citosol/enzimologia , Glicólise , Humanos , Membranas Intracelulares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfoglicerato Quinase/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Our previous work showed that in T98G cells, a human glioblastoma multiforme-derived cell line, the association of c-Fos to the endoplasmic reticulum (ER) and consequently, the capacity of c-Fos to activate phospholipid synthesis, is regulated by the phosphorylation state of tyrosine (tyr) residues #10 and #30 of c-Fos. The small amount of c-Fos present in quiescent cells is tyr-phosphorylated, is dissociated from the ER membranes and does not activate phospholipid synthesis. However, on induction of the cell to re-enter growth, c-Fos expression is rapidly induced, it is found dephosphorylated, associated to ER membranes and activating phospholipid synthesis (Portal et al., 2007). Herein, using in vivo and in vitro experimental strategies, we show that the kinase c-Src is capable of phosphorylating tyr residues of c-Fos whereas the phosphatase TC45 T-cell protein-tyr phosphatase (TC-PTP) dephosphorylates them, thus enabling c-Fos/ER association and activation of phospholipid synthesis. Results also suggest that the regulation of the phosphorylation/dephosphorylation cycle of c-Fos occurs at the TC-PTP level: induction of cells to re-enter growth promotes the translocation of TC45 from a nuclear to a cytoplasmic location concomitant with its activation. Activated TC45 in its turn promotes dephosphorylation of pre-formed c-Fos, enabling cells to rapidly activate phospholipid synthesis to respond to its growth demands.
Assuntos
Fosfolipídeos/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Tirosina/metabolismo , Animais , Proteína Tirosina Quinase CSK , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Cinética , Camundongos , Células NIH 3T3 , Fosforilação , Transporte Proteico , Quinases da Família srcRESUMO
This study establishes the role of P(5A)-type Cta4 ATPase in Ca(2+) sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive (45)Ca(2+)-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca(2+)-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4(+) gene. Furthermore, a disruption of cta4(+) resulted in 6-fold increase of intracellular Ca(2+) levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca(2+) pumping. These data provide compelling biochemical evidence for a P(5A)-type Cta4 ATPase as an essential component of Ca(2+) transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Trifosfato de Adenosina/farmacologia , Transporte Biológico/efeitos dos fármacos , Calcineurina/metabolismo , Inibidores de Calcineurina , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Deleção de Genes , Glicosilação/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Membranas Intracelulares/enzimologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (â¼1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complexo de Golgi/enzimologia , Membranas Intracelulares/enzimologia , Fígado/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Biocatálise , ATPases Transportadoras de Cobre , Fosforilação , Sus scrofa , Fatores de TempoRESUMO
Haloperidol (HAL), olanzapine (OLZ), and sulpiride (SULP) are antipsychotic drugs widely used in the pharmacotherapy of psychopathological symptoms observed in schizophrenia or mood-related psychotic symptoms in affective disorders. Here, we tested the in vitro effects of different concentrations of a typical (HAL) and two atypical (OLZ and SULP) antipsychotic drugs on ectonucleotidase activities from zebrafish brain membranes. HAL inhibited ATP (28.9%) and ADP (26.5%) hydrolysis only at 250 microM. OLZ decreased ATPase activity at all concentrations tested (23.8-60.7%). SULP did not promote significant changes on ATP hydrolysis but inhibited ADP hydrolysis at 250 microM (25.6%). All drugs tested, HAL, OLZ, and SULP, did not promote any significant changes on 5'-nucleotidase activity in the brain membranes of zebrafish. These findings demonstrated that antipsychotic drugs could inhibit NTPDase activities whereas did not change 5'-nucleotidase. Such modulation can alter the adenosine levels, since the ectonucleotidase pathway is an important source of extracellular adenosine. Thus, it is possible to suggest that changes promoted by antipsychotic drugs in the bilayer membrane could alter the NTPDase activities, modulating extracellular ATP and adenosine levels.
Assuntos
5'-Nucleotidase/metabolismo , Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Nucleosídeo-Trifosfatase/metabolismo , 5'-Nucleotidase/antagonistas & inibidores , Animais , Antipsicóticos/farmacologia , Benzodiazepinas/farmacologia , Encéfalo/enzimologia , Relação Dose-Resposta a Droga , Feminino , Haloperidol/farmacologia , Hidrólise , Membranas Intracelulares/enzimologia , Masculino , Nucleosídeo-Trifosfatase/antagonistas & inibidores , Olanzapina , Sulpirida/farmacologia , Peixe-ZebraRESUMO
Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2alpha kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2alpha at Ser51. It also phosphorylates the highly unusual form of eIF2alpha found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2alpha, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2alpha kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2alpha kinase described in unicellular eukaryotes.
Assuntos
Membrana Celular/enzimologia , Flagelos/enzimologia , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/enzimologia , eIF-2 Quinase/metabolismo , Sequência de Aminoácidos , Animais , Endossomos/enzimologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Glicosilação , Humanos , Membranas Intracelulares/enzimologia , Estágios do Ciclo de Vida , Mamíferos , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , eIF-2 Quinase/químicaRESUMO
We have isolated from rat cerebral cortex an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, which modulates glutamatergic N-methyl-d-aspartate (NMDA) receptor. This endogenous factor allosterically decreases [(3)H]dizocilpine binding to NMDA receptor, most likely acting as a weak channel blocker. In the present study we investigated whether endobain E is present in the cerebral cortex of rats subjected to ischemia and modulates NMDA receptor exposed to the same conditions. Ischemia-reperfusion was carried out by bilateral occlusion of common carotid arteries followed by a 15-min reperfusion period. Elution profile of brain soluble fraction showed that endobain E is present in cerebral cortex of ischemia-reperfusion rats. On assaying its effect on synaptosomal membrane Na(+), K(+)-ATPase activity and [(3)H]dizocilpine binding to cerebral cortex membranes prepared from animals without treatment, it was found that the endogenous modulator isolated from ischemia-reperfusion rats was able to inhibit both enzyme activity and ligand binding. On the other hand, endobain E prepared from rats without treatment also decreased binding to cerebral cortex or hippocampal membranes obtained from animals exposed to ischemia-reperfusion. Since ischemia decreases tissue pH and NMDA receptor activity varies according to proton concentration, pH influence on endobain E effect was tested. Endobain E ( approximately 80 mg original tissue) decreased [(3)H]dizocilpine binding 25% at pH 7.4 or 8.0 but 90% at pH 6.5. These results demonstrate that endobain E is present and also able to modulate NMDA receptor in the short-term period that follows cerebral ischemia and that its effect depends on proton concentration, suggesting greater NMDA receptor modulation by endobain E at low pH, typical of ischemic tissues.
Assuntos
Isquemia Encefálica/metabolismo , Córtex Cerebral/metabolismo , Ouabaína/análogos & derivados , Receptores de N-Metil-D-Aspartato/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/enzimologia , Córtex Cerebral/enzimologia , Modelos Animais de Doenças , Maleato de Dizocilpina/metabolismo , Concentração de Íons de Hidrogênio , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Masculino , Ouabaína/metabolismo , Ligação Proteica , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/etiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
Male rats exposed for 21 days to high altitude (4,340 m) responded with arrest of weight gain and increased hematocrit and testosterone levels. High altitude significantly (58%) increased heart mitochondrial nitric oxide (NO) synthase (mtNOS) activity, whereas heart cytosolic endothelial NOS (eNOS) and liver mtNOS were not affected. Western blot analysis found heart mitochondria reacting only with anti-inducible NOS (iNOS) antibodies, whereas the postmitochondrial fraction reacted with anti-iNOS and anti-eNOS antibodies. In vitro-measured NOS activities allowed the estimation of cardiomyocyte capacity for NO production, a value that increased from 57% (sea level) to 79 nmol NO.min(-1).g heart(-1) (4,340 m). The contribution of mtNOS to total cell NO production increased from 62% (sea level) to 71% (4340 m). Heart mtNOS activity showed a linear relationship with hematocrit and a biphasic quadratic association with estradiol and testosterone. Multivariate analysis showed that exposure to high altitude linearly associates with hematocrit and heart mtNOS activity, and that testosterone-to-estradiol ratio and heart weight were not linearly associated with mtNOS activity. We conclude that high altitude triggers a physiological adaptive response that upregulates heart mtNOS activity and is associated in an opposed manner with the serum levels of testosterone and estradiol.
Assuntos
Altitude , Mitocôndrias Cardíacas/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Peso Corporal , Estradiol/sangue , Membranas Intracelulares/enzimologia , Masculino , Ratos , Ratos Sprague-Dawley , Análise de Regressão , Partículas Submitocôndricas/enzimologiaRESUMO
GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.
Assuntos
Detergentes/química , Gangliosídeos/biossíntese , Gangliosídeos/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/química , Membranas Intracelulares/química , Animais , Células CHO/química , Células CHO/enzimologia , Células CHO/metabolismo , Extratos Celulares/química , Linhagem Celular , Membrana Celular/química , Cricetinae , Complexo de Golgi/enzimologia , Humanos , Membranas Intracelulares/enzimologia , Microdomínios da Membrana/química , Octoxinol/metabolismo , Sialiltransferases/biossínteseRESUMO
Maize root tonoplasts are able to accumulate Ca(2+) using the energy derived from the H(+) gradient formed during PP(i) hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca(2+) accumulated by tonoplast. Two apparently different K(s) values for Ca(2+) with values of 0.36 and 4.70 microM were detected when oxalate was included in the medium and the free Ca(2+) concentration in the medium was buffered with the use of EGTA. Binding of Ca(2+) to the outer surface of tonoplasts inhibits the outflow of Ca(2+) previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 microM Ca(2+). Thapsigargin, a specific inhibitor of Ca(2+)-ATPase, inhibits the Ca(2+) uptake driven by H(+) gradient but does not inhibit the hydrolysis of PP(i) nor the formation of a H(+) gradient.
Assuntos
Antiporters/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions , Difosfatos/metabolismo , Inibidores Enzimáticos/farmacologia , Tapsigargina/farmacologia , Zea mays/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Membranas Intracelulares/enzimologia , Transporte de Íons/efeitos dos fármacos , Cinética , Oxalatos/farmacologiaRESUMO
Nucleotides, e.g. ATP and ADP, are important signaling molecules, which elicit several biological responses. The degradation of nucleotides is catalyzed by a family of enzymes called NTPDases (nucleoside triphosphate diphosphohydrolases). The present study reports the enzymatic properties of a NTPDase (CD39, apyrase, ATP diphosphohydrolase) in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in a pH range of 7.5-8.0 in the presence of Ca(2+) (5 mM). The enzyme displayed a maximal activity for ATP and ADP hydrolysis at 37 degrees C. It was able to hydrolyze purine and pyrimidine nucleosides 5'-di and triphosphates, being insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (0.1 mM). A significant inhibition of ATP and ADP hydrolysis (68% and 34%, respectively) was observed in the presence of 20 mM sodium azide, used as a possible inhibitor of ATP diphosphohydrolase. Levamisole (1 mM) and tetramisole (1 mM), specific inhibitors of alkaline phosphatase and P1, P(5)-di (adenosine 5'-) pentaphosphate, an inhibitor of adenylate kinase did not alter the enzyme activity. The presence of a NTPDase in brain membranes of zebrafish may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in central nervous system of this specie.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Encéfalo/enzimologia , Membranas Intracelulares/enzimologia , Peixe-Zebra , Animais , Hidrólise , Nucleosídeo-Trifosfatase , Especificidade por SubstratoRESUMO
The effect of O(2) concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O(2)(-) production was determined in rat liver, brain, and kidney submitochondrial membranes. The K(mO(2)) for mtNOS were 40, 73, and 37 microM O(2) and the V(max) were 0.51, 0.49, and 0.42 nmol NO/minmg protein for liver, brain, and kidney mitochondria, respectively. The rates of O(2)(-) production, 0.5-12.8 nmol O(2)(-)/minmg protein, depended on O(2) concentration up to 1.1mM O(2). Intramitochondrial NO, O(2)(-), and ONOO(-) steady-state concentrations were calculated for the physiological level of 20 microM O(2); they were 20-39 nM NO, 0.17-0.33 pM O(2)(-), and 0.6-2.2 nM ONOO(-) for the three organs. These levels establish O(2)/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.
Assuntos
Mitocôndrias/enzimologia , Óxido Nítrico Sintase/metabolismo , Oxigênio/farmacologia , Animais , Encéfalo/enzimologia , Relação Dose-Resposta a Droga , Feminino , Membranas Intracelulares/enzimologia , Rim/enzimologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , NADP/metabolismo , Óxido Nítrico/biossíntese , Oxirredução , Ácido Peroxinitroso/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismoRESUMO
A significant portion of brain phosphatidylethanolamine (PE) is synthesized by a pathway involving the mitochondrial enzyme phosphatidylserine decarboxylase (PSDC), in a process by which phosphatidylserine (PS) is transferred from the endoplasmic reticulum to mitochondria. Aging changes the fatty acid composition of brain phospholipids, PS and PE being the most affected. The present study was carried out to determine PSDC activity in cerebral cortex (CC) and cerebellum (CRBL) mitochondrial fraction from adult (4-month-old) and aged (30-month-old) rats and to compare these activities with that found in liver. To study the effect of 22:6n-3 content on the PSDC activity, PSs from different sources were prepared: rPS (from bovine retina, containing 36 mol % of 22:6n-3); adPS (from adult rat CC microsomal membranes, with 25 mole % 22:6n-3 content) and agPS (from aged rat CC microsomal membranes, with 21 mole % 22:6n-3 content). For aged CC PSDC, the preferred substrate was agPS (the physiological substrate for aged animals), whereas in adult CC PSDC the substrate preference was inverse (rPS > adPS > agPS). Furthermore, CRBL PSDC does not show any substrate preference based on 22:6n-3 content. CRBL PSDC activity in aged membranes using agPS as substrate is lower than PSDC activity in adult membranes in the presence of adPS. These results indicate that under physiological conditions, cerebellar PSDC is inhibited during aging. Liver PSDC activity showed the same substrate preference in adult and aged rats as adult CC PSDC. These findings lead us to conclude that PSDC activity has a differential tissue-dependent substrate preference characteristic of the aging process.
Assuntos
Envelhecimento/metabolismo , Carboxiliases/metabolismo , Sistema Nervoso Central/enzimologia , Membranas Intracelulares/enzimologia , Mitocôndrias/enzimologia , Neurônios/enzimologia , Envelhecimento/patologia , Animais , Bovinos , Sistema Nervoso Central/fisiopatologia , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos Wistar , Retina/enzimologia , Frações SubcelularesRESUMO
The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Complexo de Golgi/enzimologia , Membranas Intracelulares/enzimologia , Pisum sativum/enzimologia , Tapsigargina/farmacologia , Trifosfato de Adenosina/metabolismo , Brassica/efeitos dos fármacos , Brassica/fisiologia , Cádmio/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/efeitos dos fármacos , Calmodulina/farmacologia , Cobalto/metabolismo , Cobre/farmacologia , Fucosiltransferases/metabolismo , Complexo de Golgi/efeitos dos fármacos , Indóis/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Ferro/metabolismo , Cinética , Manganês/farmacologia , Pisum sativum/efeitos dos fármacos , Vanadatos/farmacologiaRESUMO
Gamma-linolenic acid (GLA) is known to be an inhibitor of Walker 256 tumour growth in vivo and causes changes in both mitochondrial structure and cellular metabolism. The aim of the present study was to investigate in greater detail the changes in energy metabolism and ultrastructure induced by GLA in this tumour model. A diet containing 5.5% GLA, which is sufficient to cause a 45% decrease in tumour growth, was found to almost double the triacylglycerol (TAG) content of the tumour and to increase the quantity of 20:3 n-6, 20:4 n-6, 22:4 n-6 and 22:5 n-6 in the TAG fraction as determined by gas chromatography-mass spectrometry (GCMS) analysis. Morphometric analysis of the tumour by electron microscopy confirmed this increase in TAG content, identifying a doubling of lipid droplet content in the GLA dietary group. The surface density of mitochondrial cristae was reduced, along with a reduction in the number of contact sites (CS) and matrix granules. These three parameters are likely indicators of a reduction in mitochondrial metabolic activity. Measurement of hexokinase activity identified that much of the total hexokinase activity was in the mitochondrially bound form (66.5%) in the control tumour and that GLA caused a decrease in the amount of enzyme in the bound form (39.3%). The fatty acyl chain composition of the tumour mitochondrial subfractions, outer membranes (OM), CSs and inner membranes (IM) was determined by GCMS. All subfractions showed considerable increases in 20:3 n-6 and decreases in 18:1 n-9, 18:2 n-6 and 22:6 n-3, when exposed to GLA diet. These changes were reflected in a large increase in the n-6/n-3 ratio in the GLA OM vs. the control OM, 21.299 vs. 6.747, respectively. The maximal activity of OM carnitine palmitoyltransferase I (CPT I) was found to be decreased by 61.6% in the GLA diet group. This was accompanied by a decrease in malonyl CoA sensitivity and a decrease in affinity for 16:0 CoA substrate. Such changes in CPT I may be the cause of cytoplasmic acyl CoA accumulation seen in this tumour model. These effects, together with previously reported increases in lipid peroxidation, lead to the conclusion that GLA may cause inhibition of tumour cell growth through separate but interlinked pathways, all of which eventually lead to apoptosis and a decrease in tumour development. The influence of mitochondrial OM fatty acyl chain composition upon two important enzymes of energy metabolism, hexokinase and CPT I, both of which have been linked to apoptosis, is of considerable importance for future studies on fatty acid-induced cell death.
Assuntos
Carcinoma 256 de Walker/enzimologia , Carnitina O-Palmitoiltransferase/metabolismo , Hexoquinase/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ácido gama-Linolênico/farmacologia , Animais , Carcinoma 256 de Walker/ultraestrutura , Dieta , Cromatografia Gasosa-Espectrometria de Massas , Membranas Intracelulares/enzimologia , Masculino , Microscopia Eletrônica , Mitocôndrias/enzimologia , Ligação Proteica , Ratos , Ratos Wistar , Ácido gama-Linolênico/administração & dosagemRESUMO
We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.
Assuntos
ATPase de Ca(2+) e Mg(2+)/análise , Histocitoquímica , Membranas Intracelulares/enzimologia , Peroxissomos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , ATPase de Ca(2+) e Mg(2+)/metabolismo , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Ácidos Fíbricos , Fibroblastos/enzimologia , Humanos , Fígado/ultraestrutura , Mitocôndrias/enzimologia , Proliferadores de Peroxissomos/farmacologia , Ratos , Ratos Sprague-Dawley , Pele/ultraestrutura , Especificidade por SubstratoRESUMO
Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.
Assuntos
Encéfalo/enzimologia , Metaloendopeptidases/metabolismo , Neuroglia/enzimologia , Neurônios/enzimologia , Neuropeptídeos/metabolismo , Animais , Encéfalo/ultraestrutura , Compartimento Celular/fisiologia , Estruturas do Núcleo Celular/enzimologia , Estruturas do Núcleo Celular/ultraestrutura , Córtex Cerebelar/enzimologia , Córtex Cerebelar/ultraestrutura , Córtex Cerebral/enzimologia , Córtex Cerebral/ultraestrutura , Citoesqueleto/enzimologia , Citoesqueleto/ultraestrutura , Dendritos/enzimologia , Dendritos/ultraestrutura , Imuno-Histoquímica , Membranas Intracelulares/enzimologia , Membranas Intracelulares/ultraestrutura , Masculino , Microscopia Eletrônica , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Organelas/enzimologia , Organelas/ultraestrutura , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Núcleo Solitário/enzimologia , Núcleo Solitário/ultraestruturaRESUMO
The superficial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase (NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic reticulum, in pre- and postsynaptic profiles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal elements.
Assuntos
Compartimento Celular/fisiologia , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Células Ganglionares da Retina/enzimologia , Colículos Superiores/enzimologia , Sinapses/enzimologia , Vias Visuais/enzimologia , Animais , Imuno-Histoquímica , Membranas Intracelulares/enzimologia , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica , Microtúbulos/enzimologia , Microtúbulos/ultraestrutura , Neurônios/ultraestrutura , Óxido Nítrico/metabolismo , Organelas/enzimologia , Organelas/ultraestrutura , Ratos , Células Ganglionares da Retina/ultraestrutura , Colículos Superiores/ultraestrutura , Sinapses/ultraestrutura , Vias Visuais/ultraestruturaRESUMO
Content of cytochromes b5 and P-450, and activities of NADPH-cytochrome c (P-450) reductase (NCR) and 7-ethoxyresorufin O-deethylase (EROD) were measured in liver microsomes prepared from two South American endemic fish, Brycon cephalus and Colossoma macropomum, from tilapia, Oreochromis niloticus, and from Swiss mice, Mus musculus, which served as a control. Strong hemoglobin binding to fish liver microsomal membranes (FLM) altered visible spectra of microsomal cytochromes. Consequently, special precautions during FLM preparation, including liver perfusion followed by repeated washing of microsomes, were required in the study of microsomal cytochromes from these fish. FLM from all fish studied here had a significantly lower content of microsomal cytochromes but a similar level of NCR and EROD activities compared to mouse liver microsomes (MLM). Strong response of the monooxygenase system in O. niloticus to water pollution was detected with both specific cytochrome P-450 content and EROD activity increasing sharply. The optical spectra of hemoglobin from B. cephalus and C. macropomum were analyzed and some differences in shape and relative extinction were observed compared to known hemoglobins.