RESUMO
The majority of DNA polymerases (DNAPs) are specialized enzymes with specific roles in DNA replication, translesion DNA synthesis (TLS), or DNA repair. The enzymatic characteristics to perform accurate DNA replication are in apparent contradiction with TLS or DNA repair abilities. For instance, replicative DNAPs incorporate nucleotides with high fidelity and processivity, whereas TLS DNAPs are low-fidelity polymerases with distributive nucleotide incorporation. Plant organelles (mitochondria and chloroplast) are replicated by family-A DNA polymerases that are both replicative and TLS DNAPs. Furthermore, plant organellar DNA polymerases from the plant model Arabidopsis thaliana (AtPOLIs) execute repair of double-stranded breaks by microhomology-mediated end-joining and perform Base Excision Repair (BER) using lyase and strand-displacement activities. AtPOLIs harbor three unique insertions in their polymerization domain that are associated with TLS, microhomology-mediated end-joining (MMEJ), strand-displacement, and lyase activities. We postulate that AtPOLIs are able to execute those different functions through the acquisition of these novel amino acid insertions, making them multifunctional enzymes able to participate in DNA replication and DNA repair.
Assuntos
Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/genética , Organelas/enzimologia , Proteínas de Plantas/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Evolução Molecular , Proteínas de Plantas/metabolismoRESUMO
BACKGROUND AND AIMS: In mature quinoa (Chenopodium quinoa) seeds, the lasting endosperm forms a micropylar cone covering the radicle. The suspensor cells lie within the centre of the cone. During the final stage of seed development, the cells of the lasting endosperm accumulate protein and lipids while the rest are crushed and disintegrated. Both the suspensor and endosperm die progressively from the innermost layers surrounding the embryo and extending towards the nucellar tissue. Ricinosomes are endoplasmic reticulum-derived organelles that accumulate both the pro-form and the mature form of cysteine endopeptidase (Cys-EP), first identified in castor bean (Ricinus communis) endosperm during germination. This study sought to identify associations between the presence of ricinosomes and programmed cell death (PCD) hallmarks in suspensor and endosperm cells predestined to die during quinoa seed development. METHODS: A structural study using light microscopy and transmission electron microscopy was performed. To detect the presence of Cys-EP, both western blot and in situ immunolocalization assays were carried out using anti-R. communis Cys-EP antibody. A TUNEL assay was used to determine DNA fragmentation. RESULTS AND CONCLUSIONS: Except for the one or two cell layers that constitute the lasting endosperm in the mature seed, ricinosomes were found in suspensor and endosperm cells. These cells were also the site of morphological abnormalities, including misshapen and fragmented nuclei, vesiculation of the cytosol, vacuole collapse and cell wall disorganization. It is proposed that, in suspensor and endosperm cells, the early detection of Cys-EP in ricinosomes predicts the occurrence of PCD during late seed development.
Assuntos
Chenopodium quinoa/citologia , Chenopodium quinoa/crescimento & desenvolvimento , Endosperma/citologia , Endosperma/crescimento & desenvolvimento , Organelas/metabolismo , Morte Celular , Núcleo Celular/metabolismo , Chenopodium quinoa/enzimologia , Chenopodium quinoa/ultraestrutura , Cisteína Endopeptidases/metabolismo , Fragmentação do DNA , Endosperma/ultraestrutura , Citometria de Fluxo , Organelas/enzimologia , Organelas/ultraestrutura , Frações Subcelulares/metabolismoRESUMO
Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP(+)/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.
Assuntos
Vias Biossintéticas/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Esteróis/biossíntese , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Animais , Membrana Celular/química , Expressão Gênica , NADP/análise , NADPH-Ferri-Hemoproteína Redutase/genética , Organelas/enzimologia , Fagocitose , Trypanosoma cruzi/químicaRESUMO
The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.
Assuntos
Leishmania/enzimologia , Proteínas de Protozoários/metabolismo , Serina Proteases/metabolismo , Animais , Anticorpos Antiprotozoários/imunologia , Cátions Bivalentes/metabolismo , Coenzimas/metabolismo , Estabilidade Enzimática , Hemoglobinas/metabolismo , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Peso Molecular , Organelas/enzimologia , Ovalbumina/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Coelhos , Serina Proteases/química , Serina Proteases/isolamento & purificação , Soroalbumina Bovina/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB(4) and LTC(4) synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB(4) and LTC(4). Consistent with MCP-1/CCL-2 role in ox-LDL-induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies--specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling--that may amplify inflammatory mediator production in atherosclerosis.
Assuntos
Quimiocina CCL2/metabolismo , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Leucotrienos/biossíntese , Lipídeos/química , Lipoproteínas LDL/farmacologia , Organelas/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Proteínas de Transporte/metabolismo , Compartimento Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Espumosas/citologia , Células Espumosas/enzimologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organelas/efeitos dos fármacos , Organelas/enzimologia , Perilipina-2 , Receptores CCR2/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.
Assuntos
Lisossomos/ultraestrutura , Organelas/ultraestrutura , Trypanosoma cruzi/ultraestrutura , Animais , Carboxipeptidases/análise , Cisteína Endopeptidases/análise , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Estágios do Ciclo de Vida , Lisossomos/enzimologia , Microscopia Eletrônica de Transmissão , Organelas/enzimologia , ATPases Translocadoras de Prótons/análise , Proteínas de Protozoários/análise , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Giardia lamblia is a protozoan parasite with many characteristics common among eukaryotic cells, but lacking other features found in most eukaryotes. Cardiolipin is a phospholipid located exclusively in energy transducing membranes and it was identified in mitochondria, bacteria, hydrogenosomes and chloroplasts. In eukaryotes, cardiolipin is the only lipid that is synthesized in the mitochondria. Biochemical procedures (TLC, HPLC) and fluorescent tools (NAO) were applied in order to search for cardiolipin in G. lamblia. In addition, BLAST searches were used to find homologs of enzymes that participate in the cardiolipin synthesis. Cardiolipin synthase was searched in the Giardia genome, using Saccharomyces cerevisiae and Mycoplasma penetrans sequences as bait. However, a good match to G. lamblia related proteins was not found. Here we show that mitosomes of G. lamblia apparently do not contain cardiolipin, which raises the discussion for its endosymbiotic origin and for the previous proposal that Giardia mitosomes are modified mitochondria.
Assuntos
Cardiolipinas/análise , Giardia lamblia/química , Mitocôndrias/química , Sequência de Aminoácidos , Animais , Cardiolipinas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Sequência Conservada , Giardia lamblia/enzimologia , Iodo , Lipídeos/isolamento & purificação , Proteínas de Membrana/análise , Proteínas de Membrana/química , Mycoplasma penetrans/enzimologia , Organelas/química , Organelas/enzimologia , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Transferases (Outros Grupos de Fosfato Substituídos)/análise , Transferases (Outros Grupos de Fosfato Substituídos)/químicaRESUMO
Lipid bodies (lipid droplets) are emerging as dynamic organelles involved in lipid metabolism and inflammation. Increased lipid body numbers have been described in tumor cells; however, its functional significance in cancer has never been addressed. Here, we showed increased number of lipid bodies in tumor tissues from patients with adenocarcinoma of colon submitted to surgical resection when compared with an adjacent normal tissue. Accordingly, increased numbers of lipid bodies were observed in human colon adenocarcinoma cell lines and in a H-rasV12-transformed intestinal epithelial cell line (IEC-6 H-rasV12) compared with nontransformed IEC-6 cells. The functions of lipid bodies in eicosanoid synthesis in cancer cells were investigated. CACO-2 cells have increased expression of cyclooxygenase-2 (COX-2) when compared with IEC-6 cells. We showed by immunolocalization that, in addition to perinuclear stain, COX-2 and prostaglandin E (PGE) synthase present punctate cytoplasmic localizations that were concordant with adipose differentiation-related protein-labeled lipid bodies. The colocalization of COX-2 at lipid bodies was confirmed by immunoblot of subcellular fractionated cells. Direct localization of PGE(2) at its synthesis locale showed that lipid bodies are sources of eicosanoids in the transformed colon cancer cells. Treatment with either aspirin or the fatty acid synthase inhibitor C75 significantly reduced the number of lipid bodies and PGE(2) production in CACO-2 and in IEC-6 H-rasV12 cells with effects in cell proliferation. Together, our results showed that lipid bodies in colon cancer cells are dynamic and functional active organelles centrally involved in PGE(2) synthesis and may potentially have implications in the pathogenesis of adenocarcinoma of colon.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Metabolismo dos Lipídeos , Organelas/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Adenocarcinoma/enzimologia , Aspirina/farmacologia , Células CACO-2 , Processos de Crescimento Celular/fisiologia , Neoplasias do Colo/enzimologia , Células HCT116 , Células HT29 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Organelas/enzimologia , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
Trypanosoma cruzi, the etiologic agent of Chagas disease, has an unusual ATP-dependent hexokinase (TcHK) that is not affected by D-glucose 6-phosphate, but is non-competitively inhibited by inorganic pyrophosphate (PP(i)), suggesting a heterotropic modulator effect. In a previous study we identified a novel family of bisphosphonates, metabolically stable analogs of PP(i), which are potent and selective inhibitors of TcHK as well as the proliferation of the clinically relevant intracellular amastigote form of the parasite in vitro (Hudock, M. P., Sanz-Rodriguez, C. E., Song, Y., Chan, J. M., Zhang, Y., Odeh, S., Kosztowski, T., Leon-Rossell, A., Concepcion, J. L., Yardley, V., Croft, S. L., Urbina, J. A., and Oldfield, E. (2006) J. Med. Chem. 49, 215-223). In this work, we report a detailed kinetic analysis of the effects of three of these bisphosphonates on homogeneous TcHK, as well as on the enzyme in purified intact glycosomes, peroxisome-like organelles that contain most of the glycolytic pathway enzymes in this organism. We also investigated the effects of the same compounds on glucose consumption by intact and digitonin-permeabilized T. cruzi epimastigotes, and on the growth of such cells in liver-infusion tryptose medium. The bisphosphonates investigated were several orders of magnitude more active than PP(i) as non-competitive or mixed inhibitors of TcHK and blocked the use of glucose by the epimastigotes, inducing a metabolic shift toward the use of amino acids as carbon and energy sources. Furthermore, there was a significant correlation between the IC(50) values for TcHK inhibition and those for epimastigote growth inhibition for the 12 most potent compounds of this series. Finally, these bisphosphonates did not affect the sterol composition of the treated cells, indicating that they do not act as inhibitors of farnesyl diphosphate synthase. Taken together, our results suggest that these novel bisphosphonates act primarily as specific inhibitors of TcHK and may represent a novel class of selective anti-T. cruzi agents.
Assuntos
Difosfonatos/farmacologia , Inibidores Enzimáticos/farmacologia , Hexoquinase/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma cruzi/enzimologia , Animais , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Difosfatos/metabolismo , Difosfonatos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Geraniltranstransferase/metabolismo , Glucose-6-Fosfato/metabolismo , Hexoquinase/metabolismo , Humanos , Cinética , Organelas/enzimologia , Proteínas de Protozoários/metabolismo , Esteróis/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
José Ruiz-Herrera's discovery that chitin microfibrils could be made by a fungal extract paved the way for elucidating the intracellular location of chitin synthetase. In collaboration with Charles Bracker, chitosomes were identified as the major reservoir of chitin synthetase in fungi. Unique in size, buoyant density, and membrane thickness, chitosomes were found in a wide range of fungi. Their reversible dissociation into 16S subunits is another unique property of chitosomes. These 16S subunits are the smallest molecular entities known to retain chitin synthetase activity. Further dissociation leads to complete loss of activity. From studies with secretory mutants, yeast researchers concluded that chitosomes were components of the endocytosis pathway. However, key structural and enzymatic characteristics argue in favor of the chitosome being poised for exocytotic delivery rather than endocytotic recycling. The chitosome represents the main vehicle for delivering chitin synthetase to the cell surface. An immediate challenge is to elucidate chitosome ontogeny and the role of proteins encoded by the reported chitin synthetase genes in the structure or function of chitosomes. The ultimate challenge would be to understand how the chitosome integrates with the cell surface to construct the organized microfibrillar skeleton of the fungal cell wall.
Assuntos
Quitina Sintase/metabolismo , Leveduras/enzimologia , Quitina/biossíntese , Endocitose , Organelas/enzimologia , Organelas/ultraestrutura , Leveduras/ultraestruturaRESUMO
The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).
Assuntos
Via de Pentose Fosfato , Trypanosoma cruzi/metabolismo , Aldose-Cetose Isomerases/metabolismo , Animais , Carboidratos Epimerases/metabolismo , Dióxido de Carbono/metabolismo , Radioisótopos de Carbono/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Citoplasma/enzimologia , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Azul de Metileno/farmacologia , Organelas/enzimologia , Estresse Oxidativo , Via de Pentose Fosfato/efeitos dos fármacos , Fosfogluconato Desidrogenase/metabolismo , Transaldolase/metabolismo , Transcetolase/metabolismo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The capacity for 1-kestose uptake into the vacuole of fructan storing Jerusalem artichoke tubers was investigated. 1-kestose serves both as building block for fructan initiation and as a fructose donor for chain elongation. Tonoplast vesicles were isolated from actively storing tubers, and their vesicles were capable of transporting sucrose in a manner indicative of a sucrose/H(+) antiport. Under similar conditions, 1-kestose was not taken up by vesicles energized by either a pH jump or in the presence of ATP. When added together at 2 mM, sucrose uptake was not affected by the presence of 1-kestose. The data argues against the possible synthesis of 1-kestose in the cytosol and subsequent transport to the vacuole. The data also presents definite evidence for the existence a mechanism for sucrose accumulation in fructan storing vacuoles.
Assuntos
Helianthus/metabolismo , Organelas/metabolismo , Trissacarídeos/metabolismo , Transporte Biológico , Helianthus/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Fluorescência , Organelas/enzimologia , Sacarose/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
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
Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D-galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high-affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electron-lucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1.6-dip. Larger mbv possesses high-affinity sites for gal on their membrane.
Assuntos
Epididimo/enzimologia , beta-Galactosidase/metabolismo , Animais , Líquidos Corporais/enzimologia , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Fluorometria , Coloide de Ouro , Masculino , Organelas/enzimologia , Organelas/ultraestrutura , Ratos , beta-Galactosidase/ultraestruturaRESUMO
Glucosephosphate isomerase (PGI; EC 5.3.1.9) of Trypanosoma cruzi epimastigotes was found in about the same proportion in the glycosome and the cytosol. This subcellular distribution is similar to that of Leishmania mexicana, but contrasts with that of T. brucei bloodstream form, where the enzyme is essentially restricted to the glycosome. Glucosephosphate isomerase was highly purified from a glycosome-enriched fraction and to about 70% purity from the soluble extract. Both enzymes displayed Michaelis-Menten-Henri kinetics. Km values for fructose 6-phosphate were 0.125 +/- 0.07 and 0.80 +/- 0.10 mM for the glycosomal and the cytosolic PGIs, respectively. Erythrose-4-phosphate, 6-phosphogluconate and mannose-6-phosphate were inhibitors for both PGIs. Phosphogluconate and erythrose phosphate showed higher affinity for cytosolic PGI than for glycosomal PGI, by 2.5- and 4-fold respectively. The PGIs differed slightly in their isoelectric point (7.1 +/- 0.15 and 7.5 +/- 0.12) and optimum pH range. Both PGIs also differed in their chromatographic properties (ion-exchange and phenyl Sepharose), indicating a difference in charge and hydrophobicity, with the glycosomal enzyme being more hydrophobic. The molecular mass of both PGIs was 186,000 +/- 9000 Da, which is higher than that of other known PGIs, including those from T. brucei and other trypanosomatids. The molecular mass of the subunit, 63 kDa, is similar to that of PGIs from other sources. It appears that PGIs from T. cruzi are trimeric, in contrast with all other known PGIs which are dimeric.
Assuntos
Glucose-6-Fosfato Isomerase/isolamento & purificação , Glucose-6-Fosfato Isomerase/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Western Blotting , Citosol/enzimologia , Digitonina/química , Digitonina/farmacologia , Glucose-6-Fosfato Isomerase/química , Ponto Isoelétrico , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Peso Molecular , Organelas/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Frações Subcelulares , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Three molecular forms of serine hydroxymethyltransferase (SHMT) have been detected in choanomastigotes of Crithidia fasciculata by DEAE-cellulose chromatography. The three isoforms (named SHMT I, II, and III) presented small differences in charge and molecular weight. Digitonin treatment of intact cells suggested that SHMT III is cytosolic, whereas the other two isoforms are particle bound, one being mitochondrial (SHMT I) and the other one very likely glycosomal (SHMT II). The three SHMT isoforms were purified to homogeneity, and their physicochemical and kinetic properties studied. Determination of their native and subunit molecular masses revealed that all of them have a tetrameric structure. The three isoforms were shown to be PLP-dependent enzymes after L-cysteine and hydroxylamine hydrochloride treatments. They showed similar pH optima, bimodal kinetics for L-serine and Michaelis-Menten kinetics for THF.
Assuntos
Crithidia fasciculata/enzimologia , Glicina Hidroximetiltransferase/isolamento & purificação , Animais , Compartimento Celular , Citosol/enzimologia , Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/metabolismo , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Mitocôndrias/enzimologia , Peso Molecular , Organelas/enzimologia , Fosfato de Piridoxal/metabolismo , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
The subcellular localization of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the first committed step of the mevalonate pathway, was investigated in Trypanosoma cruzi epimastigotes using well-established cell fractionation procedures. It was found that ca. 80% of the activity of the enzyme was associated with the glycosomes, microbody-like organelles unique to kinetoplastid protozoa which contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity associated with microsomes. The glycosome-associated enzyme is not membrane-bound as it was recovered quantitatively in the aqueous phase of the biphasic system formed by Triton X-114 at 30 degrees C. Studies with digitonin-permeabilized intact epimastigotes demonstrated the presence of two pools of soluble HMG-CoA reductase in these cells, associated to the cytoplasmic and glycosomal compartments. Steady-state kinetic studies of the glycosome-associated enzyme indicated classical Michaelis-Menten behavior with Km,app (HMG-CoA) 28 +/- 3 microM, Km,app (NADPH) 37 +/- 4 microM, and Vm,app 3.9 +/- 0.2 nmol/min mg protein; the transition-state analog lovastatin behaved as a competitive inhibitor with respect to HMG-CoA with Kis 23 nM and a noncompetitive inhibitor toward NADPH with Kii 29 nM. The results are in complete agreement with recent gene cloning and expression studies which showed that T. cruzi HMG-CoA reductase lacks the NH2-terminal membrane-spanning sequence. This is the first demonstration of a soluble eukaryotic HMG-CoA reductase and also the first report on the presence of an enzyme of the isoprenoid biosynthesis pathway in glycosomes.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Fracionamento Celular , Digitonina , Glicólise , Hidroximetilglutaril-CoA Redutases/isolamento & purificação , Cinética , Organelas/enzimologia , Solubilidade , Frações Subcelulares/enzimologia , Trypanosoma cruzi/metabolismoRESUMO
Biosynthetic studies using both [14C]- and [32P]-labelled substrates and a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes. Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway [acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol-3-phosphate (G3P)] when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH. However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and lyso-phosphatidic acid acyltransferase activities were not found inside glycosomes. The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments.
Assuntos
Alquil e Aril Transferases , Fosfato de Di-Hidroxiacetona/biossíntese , Leishmania mexicana/metabolismo , Éteres Fosfolipídicos/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Animais , Coenzima A Ligases/metabolismo , Fosfato de Di-Hidroxiacetona/química , Ativação Enzimática , Glicerofosfatos/metabolismo , Leishmania mexicana/enzimologia , Organelas/enzimologia , Organelas/metabolismo , Palmitoil Coenzima A/metabolismo , Éteres Fosfolipídicos/química , Frações Subcelulares/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Transferases/metabolismo , Trypanosoma brucei brucei/enzimologiaRESUMO
Peroxisomicine is a toxic compound isolated from plants of the genus Karwinskia (Rhamnaceae). This toxin produces irreversible and selective damage to the peroxisomes of yeast cells in vivo. Peroxisomicine also inhibits catalase activity in vitro, when using purified enzyme. This paper reports on the effect of peroxisomicine on liver catalase in tissue fragments, in situ, as well as in mice intoxicated with peroxisomicine, in vivo. The catalase activity was determined by biochemical and histochemical methods. In contrast with the reported findings in vitro, the results demonstrate that there is no inhibition of the activity of tissue catalase, and suggest that catalase in situ and in vivo is protected against the inhibitory effect of peroxisomicine by an unknown factor.