RESUMO
Streptococcus uberis is a worldwide pathogen that causes intramammary infections in dairy cattle. Because virulence factors determining the pathogenicity of S. uberis have not been clearly identified so far, a commercial vaccine is not yet available. Different S. uberis strains have the ability to form biofilm in vitro, although the association of this kind of growth with the development of mastitis is unknown. The objective of this study was to evaluate the potential use as vaccine antigens of proteins from S. uberis biofilms, previously identified by proteomic and immunological analyses. The capability of eliciting a protective immune response by targeted candidates was assayed on a murine model. Sera from rabbits immunized with S. uberis biofilm preparations and a convalescent cow intra-mammary infected with S. uberis were probed against cell wall proteins from biofilm and planktonic cells previously separated by two-dimensional gel electrophoresis. Using rabbit immunized serum, two proteins were found to be up-regulated in biofilm cells as compared to planktonic cells; when serum from the convalescent cow was used, up to sixteen biofilm proteins were detected. From these proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase (FBA), and elongation factor Ts (EFTs) were chosen to be tested as vaccine antigen candidates. For this purpose, different groups of mice were immunized with the three recombinant-expressed proteins (each one formulated separately in a vaccine), and thereafter intraperitoneally challenged with S. uberis. The three proteins induced specific IgG antibodies, but a significant reduction of mortality was only observed in the groups of mice vaccinated with FBA or EFTs. These results suggest that FBA and EFTs might be considered as strong antigenic candidates for a vaccine against S. uberis bovine mastitis. Moreover, this is the first study to indicate that also in S. uberis, GAPDH, FBA and EFTs, as proteins detected in both cytoplasm and cell wall fractions, can play a second function (moonlighting), the latter being particularly involved in the virulence of such a pathogen organism.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Mastite Bovina/prevenção & controle , Infecções Estreptocócicas/veterinária , Streptococcus , Animais , Anticorpos Antibacterianos/sangue , Biofilmes , Bovinos , Eletroforese em Gel Bidimensional , Feminino , Frutose-Bifosfatase/imunologia , Regulação Bacteriana da Expressão Gênica , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Alongamento de Peptídeos/imunologia , Proteômica , Coelhos , Proteínas Recombinantes/imunologia , Infecções Estreptocócicas/prevenção & controleRESUMO
Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.
Assuntos
Anisakis/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Biblioteca de Peptídeos , Alérgenos/genética , Alérgenos/imunologia , Animais , Anisaquíase/sangue , Anisaquíase/imunologia , Anisaquíase/parasitologia , Anisakis/genética , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Helmintos/genética , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/imunologia , Biblioteca Gênica , Proteínas de Helminto/genética , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologiaRESUMO
Clonorchis sinensis is a food-borne zoonotic parasite that resides in bile ducts and causes clonorchiasis, which may result in cholelithiasis, cholecystitis, hepatic fibrosis, and liver tumors. Although total excretory secretory products (ESP) of C. sinensis adults induce hepatic fibrosis in vivo in rats, the causative mechanism is not well understood. To study components of the ESP, C. sinensis culture medium was collected and analyzed using shotgun LC-MS/MS. We identified a total of 110 proteins, including glycometabolic enzymes (such as fructose-1,6-bisphosphatase (FBPase) and enolase), detoxification enzymes (such as glutamate dehydrogenase, dihydrolipoamide dehydrogenase and cathepsin B endopeptidase), and a number of RAB family proteins. To identify a potential causative agent for hepatic fibrosis, we expressed and purified a recombinant FBPase, a 1,041-bp gene product that encodes a 41.7-kDa protein with prototypical FBPase domains and that can form a tetramer with a molecular mass of 166.8 kDa. In addition, we found that FBPase is an antigen present in the ESP and in circulation. Immunofluorescence showed that FBPase localizes to the intestinal cecum and vitellarium in C. sinensis adults. Our results describe the components of the excretory secretory products from C. sinensis adult worms and suggest that FBPase may be an important antigen present in the ESP of C. sinensis and may lay the foundation for additional studies on the development of clonorchiasis-associated hepatic fibrosis.
Assuntos
Antígenos de Helmintos/análise , Clonorchis sinensis/química , Frutose-Bifosfatase/análise , Proteoma/análise , Estruturas Animais/química , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Cromatografia Líquida , Clonagem Molecular , Meios de Cultura/química , Frutose-Bifosfatase/química , Frutose-Bifosfatase/imunologia , Expressão Gênica , Humanos , Peso Molecular , Espectrometria de Massas em TandemRESUMO
Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.
Assuntos
Aminas/metabolismo , Técnicas Biossensoriais/métodos , Biotina/análogos & derivados , Extratos Hepáticos/metabolismo , Proteínas/análise , Proteínas/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Arginase/imunologia , Arginase/metabolismo , Biotina/metabolismo , Biotinilação , Eletroforese em Gel de Poliacrilamida , Frutose-Bifosfatase/imunologia , Frutose-Bifosfatase/metabolismo , Cobaias , Immunoblotting , Dados de Sequência Molecular , Ligação Proteica , Proteínas/química , Proteínas/imunologia , Ratos , Ratos WistarRESUMO
Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our findings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immunofluorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of different localization signals on FBPase for diverse metabolic functions.
Assuntos
Núcleo Celular/enzimologia , Frutose-Bifosfatase/metabolismo , Rim/enzimologia , Fígado/enzimologia , Animais , Membrana Celular/enzimologia , Imunofluorescência , Frutose-Bifosfatase/imunologia , Immunoblotting , Isoenzimas/imunologia , Isoenzimas/metabolismo , Rim/citologia , Fígado/citologia , Microscopia Confocal , Ratos , Ratos Endogâmicos , Extratos de TecidosRESUMO
The localization of fructose-1,6-bisphosphatase (Fru-1,6-Pase EC 3.1.3.11) in human alveolar epithelium was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from human liver. The immunohistochemical analysis revealed that the Fru-1,6-Pase was localized in pneumocytes II and was absent in pneumocytes I. Hypothetically Fru-1,6-Pase participating in glucose-6-phosphate synthesis from noncarbohydrate precursors increases NADPH level which is used for surfactant synthesis and for glutathione redox cycle.
Assuntos
Frutose-Bifosfatase/metabolismo , Pulmão/citologia , Pulmão/enzimologia , Animais , Frutose-Bifosfatase/imunologia , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/enzimologia , Coelhos/imunologia , Fixação de TecidosRESUMO
Several isozymes of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase have been characterized from mammalian tissues and, based on tissue origin, they are classified as liver, skeletal muscle, heart, testis, and placenta isozymes. In this paper, we examined the tissue distribution of placenta-type isozyme in rat tissues at the levels of transcription and translation. Analysis by Northern blotting showed that placenta, brain, testis, liver, kidney, and skeletal muscle expressed mRNA of placenta-type isozyme. Western blot analysis of fractions from POROS-HQ column chromatography of extracts from various rat tissues showed that proteins of placenta-type isozyme are expressed in placenta, brain, testis, liver, spleen, heart and lung, but not in kidney and skeletal muscle. An immunohistochemical study showed that, in liver, placenta-type isozyme is localized in Kupffer cells. These results indicate that isozymes of this particular enzyme may occur in particular cell types within each tissue.
Assuntos
Frutose-Bifosfatase/metabolismo , Expressão Gênica , Complexos Multienzimáticos/metabolismo , Fosfofrutoquinase-1/metabolismo , Placenta/enzimologia , Animais , Especificidade de Anticorpos , Northern Blotting , Western Blotting , Ácido Clodrônico/farmacologia , Feminino , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/imunologia , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/enzimologia , Células de Kupffer/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/imunologia , Fosfofrutoquinase-2 , Placenta/metabolismo , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
Spinach (Spinacia oleracea) cytosolic fructose-1,6-bisphosphatase (FBPase) was purified and the final preparation of protein has a specific activity of about 45 units/mg protein and a single band of molecular mass of 39 kDa. Polyclonal antibody against the protein did not cross-react with chloroplast FBPase, but showed strong cross-reactivity with all plant cytosolic FBPases tested. Studies of the FBPase expression characteristics at early stages of development demonstrated that it was controlled at both the transcriptional and translational levels, and its mRNA was detected even in etiolated cotyledons. This suggests that the expression is not light-inducible. A single transcript was detected in all spinach tissues tested. Western blot analysis revealed two protein bands in the etiolated cotyledons: one was the same size as that present in the mature leaf, and the other slightly smaller. A high enzyme activity was detected in etiolated cotyledons, especially compared to protein levels in Western blots. Expression of the cytosolic FBPase gene during leaf development showed no change in the steady-state level of mRNA, but the protein level and enzyme activity were higher in mature leaves than in young ones, suggesting that the increase in FBPase activity during development is due to an increase in protein synthesis. Young roots showed low enzyme activity, but an unexpectedly high activity was detected in old fiber roots.
Assuntos
Frutose-Bifosfatase , Spinacia oleracea/enzimologia , Spinacia oleracea/ultraestrutura , Anticorpos/metabolismo , Cotilédone/enzimologia , Citosol/enzimologia , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/imunologia , Frutose-Bifosfatase/isolamento & purificação , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Hipocótilo/enzimologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Spinacia oleracea/crescimento & desenvolvimento , Fatores de Tempo , Distribuição TecidualRESUMO
Fructose-1,6-bisphosphatase (FBPase), the key enzyme in gluconeogenesis in the yeast Saccharomyces cerevisiae, is induced when cells are grown in medium containing poor carbon sources. FBPase is targeted from the cytosol to the vacuole for degradation when glucose-starved yeast cells are replenished with fresh glucose. In this study, we report the reconstitution of the glucose-induced import of FBPase into the vacuole in semi-intact yeast cells using radiolabeled FBPase, an ATP regenerating system and cytosol. The import of FBPase was defined as the fraction of the FBPase that was sequestered inside a membrane-sealed compartment. FBPase import requires ATP hydrolysis and is stimulated by cytosolic proteins. Furthermore, the import of FBPase is a saturable process. FBPase import is low in the glucose-starved cells and is stimulated in the glucose-replenished cells. FBPase accumulates to a higher level in the pep4 cell, suggesting that FBPase is targeted to the vacuole for degradation. Indirect immunofluorescence microscopy studies demonstrate that the imported FBPase is localized to the vacuole in the permeabilized cells. Thus, the glucose-induced targeting of FBPase into the vacuole can be reproduced in our in vitro system.
Assuntos
Frutose-Bifosfatase/metabolismo , Glucose/metabolismo , Saccharomyces cerevisiae/enzimologia , Vacúolos/enzimologia , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Compartimento Celular , Frutose-Bifosfatase/imunologia , Gluconeogênese , Microscopia de FluorescênciaRESUMO
The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-)2-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense.
Assuntos
Frutose-Bifosfatase/metabolismo , Rim/enzimologia , Fígado/enzimologia , Animais , Western Blotting , Núcleo Celular/metabolismo , Frutose-Bifosfatase/imunologia , Imuno-Histoquímica , Rim/citologia , Córtex Renal/metabolismo , Glomérulos Renais/metabolismo , Medula Renal/metabolismo , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Fígado/citologia , Alça do Néfron/metabolismo , Microscopia Confocal , Ratos , Ratos Sprague-DawleyRESUMO
The effects of the monoclonal antibodies (McAbs) directed against chicken liver 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) on the structure and function of the enzyme were studied. Using chicken liver 6PF-2-K/Fru-2, 6-P2ase as antigen, 7 clones of monoclonal antibodies specifically binding with the antigen were obtained. The epitopes of the antigen recognized by the 6 McAbs localized on the fructose-2, 6-bisphosphatase domain of chicken liver 6PF-2-K/Fru-2, 6-P2ase, and the other (H2) are on the 6-phosphofructo-2-kinase domain. All of the 7 McAbs could activate the kinase activity of the bifunctional enzyme by twofold and had a similar effect on the bisphosphatase activity of the bifunctional enzyme which resulted in a fourfold increase of the bisphosphatase activity of the bifunctional enzyme. However, the McAbs did not affect the activity of the separated fructose-2, 6-bisphosphatase domain. The results suggested that the Fru-2, 6-P2ases in the bifunctional enzyme and in the separated bisphosphatase domain were in two different conformation and activity states.
Assuntos
Anticorpos Monoclonais/imunologia , Frutose-Bifosfatase/imunologia , Complexos Multienzimáticos/imunologia , Fosfofrutoquinase-1/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fosfofrutoquinase-2 , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed whether CFBPases from different phylogenetic origin contain a common epitope. To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transferase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate. The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-agarose. This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety. Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca. 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T. aestivum and T. durum), but also with homogeneous preparations of the spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes. Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common epitope through biological evolution.
Assuntos
Anticorpos Monoclonais , Cloroplastos/enzimologia , Frutose-Bifosfatase/imunologia , Triticum/enzimologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Cloroplastos/genética , Cloroplastos/imunologia , Clonagem Molecular , Reações Cruzadas , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Evolução Molecular , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/isolamento & purificação , Glutationa Transferase/genética , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Plantas/enzimologia , Plantas/genética , Plantas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Schistosoma japonicum/enzimologia , Schistosoma japonicum/genética , Especificidade da Espécie , Triticum/genética , Triticum/imunologiaRESUMO
The characterization of fructose-1,6-bisphosphatase in stage VI oocytes from the frog Caudiverbera caudiverbera, as well as the in vivo activity, is reported. The enzyme has a subunit molecular weight of approximately 43,500, has an apparent Km value of 17 microM for fructose-1,6-bisP, and is inhibited by substrate concentrations beyond 100 microM. AMP and fructose-2,6-bisP are strong inhibitors of oocyte fructose-1,6-bisphosphatase activity with Ki values of 9 and 2 microM respectively. Inhibition by AMP is cooperative with a nH value of 2.2. In vivo fructose-1,6-bisphosphatase activity was demonstrated by microinjection of [U-14C]- or [6-32P]fructose-1,6-bisP and subsequent chromatographic separation and identification of labeled products. The relevance of these findings for the metabolism of glucose in frog oocytes is discussed.
Assuntos
Anuros/fisiologia , Frutose-Bifosfatase/isolamento & purificação , Oócitos/enzimologia , Monofosfato de Adenosina/farmacologia , Animais , Frutose-Bifosfatase/efeitos dos fármacos , Frutose-Bifosfatase/imunologia , Frutose-Bifosfatase/metabolismo , Cinética , Peso Molecular , Oócitos/metabolismo , Frações Subcelulares/enzimologiaRESUMO
The cytosolic isozymes of fructose-1,6-bisphosphatase (FBPasec) and aldolase (ALDc) from germinating castor oil seed endosperm (COS) (Ricinus communis L.; cv Hale) were purified to homogeneity and final specific activities 49 and 2.8 (mumol product produced/min)/mg protein, respectively. Nondenaturing polyacrylamide gel electrophoresis of the final FBPasec preparation resolved a single protein-staining band which comigrated with FBPase activity. Two protein-staining bands of 41 and 39 kDa that occurred in an approximate 1:1 ratio were observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final FBPasec preparation. Rabbit anti-(FBPasec) immune serum immunoprecipitated the activities of FBPasec, but not that of the plastidic isozyme of FBPase from germinated COS. Immunoblot analysis utilizing affinity purified anti-(COS FBPasec) immunoglobulin G established that the 39-kDa subunit of FBP-asec did not arise via proteolytic cleavage of the 41-kDa subunit during tissue extraction and enzyme purification. However, FBPasec was susceptible to degradation by endogenous protease(s) during incubation of an acidic (pH 5.9) clarified COS extract at 25 degrees C. This proteolysis caused the production of a 32-kDa antigenic polypeptide and resulted in FBPase inactivation. Gel filtration indicated that purified FBPasec exists in at least 8 different oligomeric forms ranging in size from > 2 million to < 34 kDa. The majority of FBPasec, however, eluted as a 143-kDa heterotetramer. Sodium dodecyl sulfate gel electrophoresis of the final ALDc preparation yielded a single 40-kDa protein-staining polypeptide that cross-reacted with anti-(carrot ALDc) IgG. FBPasec copurified with ALDc through polyethylene glycol fractionation, Q-Sepharose, and phosphocellulose chromatographies, and the intensity of the fluorescence emission spectrum of ALDc was greatly reduced in the presence of COS FBPasec, but not rabbit muscle FBPase. These findings suggest that these two metabolically sequential enzymes might specifically interact in the cytosol of the highly gluconeogenic germinating COS. Our results also demonstrate that endogenous nonspecific acid phosphatase activity can interfere with the spectrophotometric assay for FBPase and can thus result in overestimations of FBPase activity in impure plant extracts.
Assuntos
Citosol/enzimologia , Frutose-Bifosfatase/isolamento & purificação , Frutose-Bifosfato Aldolase/isolamento & purificação , Plantas Tóxicas , Ricinus communis/enzimologia , Sementes/enzimologia , Ricinus communis/crescimento & desenvolvimento , Estabilidade Enzimática , Frutose-Bifosfatase/imunologia , Peso Molecular , Conformação Proteica , Sementes/crescimento & desenvolvimento , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.
Assuntos
Cloroplastos/enzimologia , Citosol/enzimologia , Fabaceae/enzimologia , Frutose-Bifosfatase/imunologia , Plantas Medicinais , Verduras/enzimologia , Sequência de Aminoácidos , Western Blotting , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
In this paper we study activation by dithiothreitol and reduced thioredoxins and deactivation by oxidized thioredoxins f of sedoheptulose-1,7-bisphosphatase. The behaviour of the enzyme when chromatographed on a thioredoxin-Sepharose column is also described. The enzyme is autoxidizable upon removal of reducing agents, and is activated when reduced by any of the thioredoxins. This mechanism may allow the regulation of the Calvin cycle upon light-dark and dark-light transitions. The formation of a stable complex between enzyme and thioredoxin could explain the inhibitory effect of high thioredoxin concentrations. The use of immunological techniques shows that sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase are poorly related immunologically.
Assuntos
Cloroplastos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Aminoácidos/análise , Cromatografia de Afinidade , Ativação Enzimática/efeitos dos fármacos , Frutose-Bifosfatase/imunologia , Imunodifusão , Cinética , Monoéster Fosfórico Hidrolases/imunologia , Plantas/enzimologia , Tiorredoxinas/farmacologiaRESUMO
The mRNA coding for rat liver fructose-1,6-bisphosphatase, which represents approx. 0.46% of total hepatic mRNA, has been purified to near homogeneity. Polysomes from rat liver were allowed to react with antibodies to rabbit anti-fructose-1,6-bisphosphatase purified by affinity chromatography. The complex was immobilized on a protein A-Sepharose column. After the removal of unabsorbed polysomes, the specific mRNA was eluted and chromatographed on an oligo(dT)-cellulose column. This method gave a 183-fold enrichment of the fructose-1,6-bisphosphatase mRNA to greater than 80% homogeneity as determined by electrophoreses of immunoprecipitated in vitro translation products on polyacrylamide slab gels in the presence of sodium dodecyl sulphate.
Assuntos
Frutose-Bifosfatase/genética , RNA Mensageiro/isolamento & purificação , Animais , Sistema Livre de Células , Frutose-Bifosfatase/imunologia , Técnicas Imunológicas , Fígado/enzimologia , Fígado/fisiologia , Polirribossomos/imunologia , Biossíntese de Proteínas , RNA Mensageiro/genética , RatosRESUMO
An indirect immunoperoxidase procedure has been used to demonstrate sites of glycolysis and gluconeogenesis in normal rat kidney and liver. In kidney, the gluconeogenic enzyme fructose 1,6-biphosphatase was restricted to the proximal tubular epithelium, while the glycolytic enzyme hexokinase predominated in more distal segments. Intense staining for the biphosphatase in proximal convoluted tubular brush borders suggests that reabsorbed substrates may be used directly at this site in renal gluconeogenesis. In view of the high phosphofructokinase and pyruvate kinase activities present in collecting ducts, their relatively low hexokinase activities and their relatively pale immunostaining for hexokinase indicate that glycolytic substrates which feed into the pathway subsequent to the initial phosphorylation step, rather than glucose, may be the major energy source for the rat renal papilla. Immunostaining in the liver was consistent with the metabolic zonation of liver parenchyma, in that glucokinase occurred mainly in perivenous regions and fructose 1,6-bisphosphatase in periportal areas. The presence of such metabolic zonation is difficult to reconcile with the widely held view that the majority of hepatic glycogen is derived directly from glucose. A model for hepatic glycogen synthesis is proposed which links the concept of parenchymal zonal heterogeneity with recent biochemical evidence concerning the 'glucose paradox' and with microscopical studies on the dynamics of glycogen deposition after refeeding.
Assuntos
Compartimento Celular , Gluconeogênese , Glicólise , Rim/enzimologia , Fígado/enzimologia , Animais , Especificidade de Anticorpos , Feminino , Frutose-Bifosfatase/análise , Frutose-Bifosfatase/imunologia , Frutose-Bifosfatase/metabolismo , Glucoquinase/imunologia , Glucoquinase/metabolismo , Hexoquinase/imunologia , Hexoquinase/metabolismo , Histocitoquímica , Soros Imunes/análise , Rim/metabolismo , Fígado/metabolismo , Masculino , Piruvato Quinase/análise , Piruvato Quinase/metabolismo , Ratos , Ratos Endogâmicos , Coloração e RotulagemRESUMO
A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes.
Assuntos
Bacillus subtilis/genética , Frutose-Bifosfatase/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Bacillus subtilis/enzimologia , Metabolismo dos Carboidratos , Reações Cruzadas , Frutose-Bifosfatase/imunologia , Gluconatos/biossíntese , Gluconatos/metabolismo , Gluconeogênese , Inositol/biossíntese , Inositol/metabolismo , Fosfotransferases/biossíntese , Esporos Bacterianos/fisiologia , Desidrogenase do Álcool de Açúcar/biossínteseRESUMO
After the addition of glucose to acetate- or ethanol-grown yeast cells a small group of selected enzymes is rapidly inactivated. This phenomenon has been called "catabolite inactivation". Among other enzymes participating in gluconeogenesis, fructose-1,6-bisphosphatase is inactivated during this catabolite inactivation process. It was shown by FUNAYAMA et al. (Eur. J. Biochem. 109, 61-66 (1980)) that the mechanism of inactivation is proteolysis. In the present paper evidence is presented that after addition of glucose a covalent conversion of the enzyme protein by phosphorylation of a serine-residue initiates its subsequent proteolysis. It is suggested that the covalent modification triggered by glucose and/or products of its catabolism renders the enzyme susceptible to proteinases and thereby initiates proteolysis of a selected enzyme without the necessity of a specific proteinase present.