RESUMO
The genome size and base composition of diploid plant species from three genera of the Casuarinaceae family were determined by flow cytometry. Casuarina glauca Sieb. ex Spring. and Gymnostoma deplancheana (Miq.) L. Johnson showed a small genome with 2C = 0.70 pg, 58.6% AT, 40.5% GC for the first species and 2C = 0.75 pg, 58.7% AT, 40.5% GC for the second. Allocasuarina verticillata (Lam.) L. Johnson had a larger genome: 2C = 1.90 pg, 59.3% AT, 41.1% GC. One haploid genome of C. glauca is therefore about 340×106 base pairs. In leaves, roots or bark of these three species, polysomaty was virtually absent: a maximum frequency of 4C nuclei of only 0.08 was found in bark of C. glauca. The genome sizes of C. glauca and G. deplancheana are among the smallest described for higher plants. Small genome size, diploidy and the absence of polysomaty are advantageous traits for facilitating molecular approaches to improvement of these actinorhizal plants and developing the study of their symbiotic interactions with Frankia.
RESUMO
Frankia isolates from nodules of the genera Casuarina (BR, S21, Thr), Allocasuarina (Allo2), and Gymnostoma (G80) were found to grow exponentially with high biomass yield and minimal sporangia formation in stirred propionate mineral medium when supplemented either with 2.4 µM palmitic acid (C16:0), pentadecanoic (C15:0), heptadecanoic (C17:0), or linoleic (C18:2, cis 9, 12) fatty acids. Strains also grew with lauric (C12:0) or myristic (C14:0) acids, but gave lower biomass yield. Stearic acid (C18:0) produced a good biomass yield, but cultures slowly accumulated sporangia; oleic acid (C18:1, cis-9) was detrimental to growth. Caprylic (C8:0) or capric (C10:0) acids proved to be prejudicial for long-term storage of Frankia strains. In experiments using labeled 1,2-dipalmitoyl phosphatidylcholine and palmitic acid, radioactivity bound rapidly to the insoluble, but solvent-extractable fraction of Frankia cells. In contrast, label from propionic acid accumulated in the cytosolic fraction. Therefore, the beneficial effect of some specific phospatidylcholines or free fatty acids on Frankia growth appears to result from their utilization as building blocks for the membrane, suggesting that membrane biosynthesis may be the limiting step for Frankia growth in unamended propionate mineral medium.
RESUMO
A major-high-molecular mass proteinase and seven latent minor proteinases were found in cell extracts and in concentrates of culture medium from Frankia sp. strain BR after nondenaturing electrophoresis in mixed gelatin-polyacrylamide gels. All of these complexes showed multicatalytic properties. Their molecular masses and their sedimentation coefficients varied from 1,300 kDa (28S) to 270 kDa (12S). The electroeluted 1,300-kDa proteinase complex dissociated into 11 low-molecular-mass proteinases (40 to 19 kDa) after sodium dodecyl sulfate activation at 30 degrees C and electrophoresis under denaturing conditions. All of these electroeluted proteinases hydrolyzed N-carbobenzoxy-Pro-Ala-Gly-Pro-4-methoxy-beta- naphthylamide, D-Val-Leu-Arg-4-methoxy-beta-naphthylamide, and Boc-Val-Pro-Arg-4-methyl-7-coumarylamide, whereas Suc-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide was cleaved only by the six lower-molecular-mass proteinases (27.5 to 19 kDa). Examination by electron microscopy of uranyl acetate-stained, electroeluted 1,300- and 650-kDa intracellular and extracellular proteinase complexes showed ring-shaped and cylindrical particles (10 to 11 nm in diameter, 15 to 16 nm long) similar to those of eukaryotic prosomes and proteasomes. Polyclonal antibodies raised against rat skeletal muscle proteasomes cross-reacted with all of the high-molecular-mass proteinase complexes and, after denaturation of the electroeluted 1,300-kDa band, with polypeptides of 35 to 38, 65, and 90 kDa. Electrophoresis of the activated cell extracts under denaturing conditions revealed 11 to 17 gelatinases from 40 to 19 kDa, including the 11 proteinases of the 1,300-kDa proteinase complex. The inhibition pattern of these proteinases is complex. Thiol-reactive compounds and 1-10-phenanthroline strongly inhibited all of the proteinases, but inhibitors against serine-type proteinases were also effective for most of them.
Assuntos
Actinomycetales/enzimologia , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Centrifugação com Gradiente de Concentração , Reações Cruzadas , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Endopeptidases/efeitos dos fármacos , Endopeptidases/imunologia , Endopeptidases/ultraestrutura , Ativação Enzimática , Gelatina/metabolismo , Substâncias Macromoleculares , Microscopia Eletrônica , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/ultraestrutura , Coloração Negativa , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.
Assuntos
Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Frankia/fisiologia , Divisão Celular/fisiologia , Meios de Cultura , Eletroforese , Frankia/crescimento & desenvolvimento , Fatores de TempoRESUMO
Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippophaë host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.
Assuntos
Actinomyces/enzimologia , Aminopeptidases/análise , Esterases/análise , Oxirredutases/análise , Actinomyces/crescimento & desenvolvimento , Citosol/enzimologia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Sensibilidade e Especificidade , Especificidade por Substrato , Terminologia como AssuntoRESUMO
The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.
Assuntos
Endopeptidases , L-Lactato Desidrogenase/isolamento & purificação , Pichia/enzimologia , Saccharomycetales/enzimologia , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Hidrólise , Cinética , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase (Citocromo) , Mitocôndrias/enzimologia , NADH Desidrogenase/análise , Fragmentos de Peptídeos/isolamento & purificação , Frações Subcelulares/enzimologiaRESUMO
The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.
Assuntos
Membrana Celular/enzimologia , Quitina Sintase/metabolismo , Glucosiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPase de Ca(2+) e Mg(2+)/metabolismo , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Concanavalina A/metabolismo , Microscopia Eletrônica , Organelas/enzimologia , Saccharomyces cerevisiae/ultraestrutura , Frações Subcelulares/enzimologia , Frações Subcelulares/ultraestruturaRESUMO
Alterations in the synthesis of proteins following exposure of Saccharomyces cerevisiae to UV light were investigated using radioactive labelling and two dimensional electrophoresis. UV-irradiation induced the synthesis of various proteins. Among them the analogue of the RecA protein of Escherichia coli (Angulo et al. 1985) and two other polypeptides (34 Kd and 35 Kd, pI 5.8) were observed in all four strains analyzed namely two DNA-repair deficient (rad-) strains: (rad6-1 and pso2-1) and their isogenic wild type RAD+ strains.
Assuntos
Proteínas Fúngicas/biossíntese , Biossíntese Peptídica , Saccharomyces cerevisiae/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Raios UltravioletaRESUMO
In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K 12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD+) strains carrying mitochondrial DNA or not (rho0). This was also true for the following DNA-repair deficient (rad-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-delta, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.
Assuntos
Escherichia coli/genética , Recombinases Rec A/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação , Raios Ultravioleta , Anticorpos , Relação Dose-Resposta à Radiação , Genótipo , Cinética , Saccharomyces cerevisiae/genética , Especificidade da EspécieRESUMO
Most of the X-prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx. 75-times higher than in the protoplast lysate). The tonoplast-bound enzyme is thermosensitive. Another heat-resistant enzyme was found in the protoplast lysate. The tonoplast-bound thermosensitive enzyme shows an apparent Km of 0.06 mM against L-alanyl-L-prolyl-p-nitroanilide while the heat-resistant enzyme shows an apparent Km of 0.4 mM against the same substrate.
Assuntos
Aminopeptidases/metabolismo , Organoides/enzimologia , Saccharomyces cerevisiae/enzimologia , Vacúolos/enzimologia , Temperatura Alta , Membranas Intracelulares/enzimologia , Cinética , Saccharomyces cerevisiae/ultraestruturaRESUMO
The subcellular distribution of five aminoacyl-tRNA synthetases from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated. To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles. The vacuole-free supernatant was subjected to sucrose density gradient centrifugation. No evidence for multimolecular associations of these enzymes was found. In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction. A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit. The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes. The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis. Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.
Assuntos
Aminoacil-tRNA Sintetases/isolamento & purificação , Fígado/enzimologia , Saccharomyces cerevisiae/enzimologia , Aminoacil-tRNA Sintetases/metabolismo , Animais , Cinética , Coelhos , Especificidade da Espécie , Esferoplastos/enzimologia , Frações Subcelulares/enzimologiaRESUMO
Disruption of yeast spheroplasts by DEAE-dextran in isoosmotic conditions allows isolation of relatively undamaged subcellular fractions from yeast. The preservation of mitochondria and vacuoles permits the simultaneous isolation of the cytosol with negligible contamination by vacuolar proteinases and therefore, virtually eliminates proteolytic artefacts.
Assuntos
Fracionamento Celular/métodos , Citosol/enzimologia , Mitocôndrias/enzimologia , Organoides/enzimologia , Saccharomyces cerevisiae/enzimologia , Vacúolos/enzimologia , Endopeptidases/isolamento & purificação , Glutamato Desidrogenase/isolamento & purificação , Saccharomyces cerevisiae/ultraestrutura , Esferoplastos/enzimologia , TemperaturaRESUMO
Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD+ yeast cells after a dose of 50 Jm-2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD+ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.
Assuntos
Reparo do DNA , Peptídeo Hidrolases/genética , Saccharomyces cerevisiae/genética , Serina Endopeptidases , Aminopeptidases/metabolismo , Carboxipeptidases/metabolismo , Cicloeximida/farmacologia , Endopeptidases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains. It is obvious that the induction of protease B activity following UV-treatment in Saccharomyces cannot be equated to the induction of the recA protein in Escherichia coli. However the correlation found between the block in mutagenic repair and the lack of UV-induction of protease B activity leads to questions on the possible role of certain protease activities in mutagenic repair in eucaryotic cells.
Assuntos
Reparo do DNA , Peptídeo Hidrolases/genética , Saccharomyces cerevisiae/genética , Serina Endopeptidases , Endopeptidases/metabolismo , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
Virtually all of the polyphosphate (PP) present in yeast protoplasts can be recovered in a crude particulate fraction if polybase-induced lysis is used for disrupting the protoplasts. This fraction contains most of the vacuoles, mitochondria and nuclei. Upon the purification of vacuoles the PP is enriched to the same extent as are the vacuolar markers. The amount of PP per vacuole is comparable to the amount of PP per protoplast. The possibility that PP is located in the cell wall is also considered. In the course of the incubation necessary for preparing protoplasts, 20% of the cellular PP is broken down. As this loss of PP occurs to the same extent in the absence of cell wall degrading enzymes, it is inferred that internal PP is metabolically degraded, no PP being located in the cell walls. It is concluded that in Saccharomyces cerevisiae most if not all of the PP is located in the vacuoles, at least under the growth conditions used.
Assuntos
Polifosfatos/isolamento & purificação , Saccharomyces cerevisiae/análise , Parede Celular/análise , Protoplastos/análise , Frações Subcelulares , Vacúolos/análiseRESUMO
The conversion of stationary and starved yeast cells into protoplasts is described. The method is rapid, simple and can be applied to a variety of stationary yeast cells. Preincubation of yeast cells in the presence of pronase was essential for effective conversion into protoplasts. Baker's yeast and seven defined yeast strains, including one "petite", were studied. All of them were efficiently transformed into protoplasts in 60 to 90 min, depending on the strain culture conditions and the age of the culture. Protoplasts may be obtained even from late-stationary cells which contain spores. Saccharomyces cerevisiae cells subjected to complete starvation conditions in water, could also be completely transformed into protoplasts, even after 48 h of starvation. Electron microscope examination of stationary protoplasts from three different yeast strains showed no evidence of a remaining cell-wall. S. cerevisiae stationary cells show a very developed vacuolar system, a number of "lipid granules" and a few altered mitochondria. Endomycopsis fibuligera and Candida tropicalis stationary protoplasts show a similar fine structure, but "lipid granules" were completely absent.