RESUMO
Time-resolved fluorescence spectroscopy was used to explore the pathway and kinetics of energy transfer in photosynthetic membrane vesicles (chromatophores) isolated from Rhodobacter (Rba.) sphaeroides cells harvested 2, 4, 6 or 24 hours after a transition from growth in high to low level illumination. As previously observed, this light intensity transition initiates the remodeling of the photosynthetic apparatus and an increase in the number of light harvesting 2 (LH2) complexes relative to light harvesting 1 (LH1) and reaction center (RC) complexes. It has generally been thought that the increase in LH2 complexes served the purpose of increasing the overall energy transmission to the RC. However, fluorescence lifetime measurements and analysis in terms of energy transfer within LH2 and between LH2 and LH1 indicate that, during the remodeling time period measured, only a portion of the additional LH2 generated are well connected to LH1 and the reaction center. The majority of the additional LH2 fluorescence decays with a lifetime comparable to that of free, unconnected LH2 complexes. The presence of large LH2-only domains has been observed by atomic force microscopy in Rba. sphaeroides chromatophores (Bahatyrova et al., Nature, 2004, 430, 1058), providing structural support for the existence of pools of partially connected LH2 complexes. These LH2-only domains represent the light-responsive antenna complement formed after a switch in growth conditions from high to low illumination, while the remaining LH2 complexes occupy membrane regions containing mixtures of LH2 and LH1-RC core complexes. The current study utilized a multi-parameter approach to explore the fluorescence spectroscopic properties related to the remodeling process, shedding light on the structure-function relationship of the photosynthetic assembles. Possible reasons for the accumulation of these largely disconnected LH2-only pools are discussed.
Assuntos
Adaptação Fisiológica , Transferência de Energia , Luz , Rhodobacter sphaeroides/química , Microscopia de Força Atômica , Rhodobacter sphaeroides/fisiologia , Espectrometria de FluorescênciaRESUMO
The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.
Assuntos
Bacterioclorofilas/metabolismo , Membrana Celular/ultraestrutura , Clorofila/análogos & derivados , Rhodobacter sphaeroides/ultraestrutura , Aerobiose , Membrana Celular/metabolismo , Oxirredução , Fotossíntese , Rhodobacter sphaeroides/metabolismo , EspectrofotometriaRESUMO
Chromatophore membrane formation was induced in low-aeration suspensions of Rhodopseudomonas sphaeroides and highly purified chromatophore preparations were isolated at various intervals between 4 and 18 h. The levels of several functional components associated with the isolated strucures were investigated. B-875, the light-harvesting bacteriochlorophyll complex associated with the reaction center, was preferentially inserted into the chromatophore membrane during the early stages of induction, and thereafter its levels reached a steady state; b- and c-type cytochromes were also maintained at essentially constant levels. In contrast, the levels of B-850, the accessory light-harvesting bacteriochlorophyll, together with its associated protein, continued to increase throughout the induction process. Increases in the levels of the major carotenoid component followed a similar course. These findings are consistent with a stepwise assembly mechanism for associated bacteriochlorophyll and protein components and suggest that separate regulatory mechanisms control the levels of functionally essential and accessory components within the membrane.
Assuntos
Cromatóforos Bacterianos/ultraestrutura , Rhodobacter sphaeroides/ultraestrutura , Aerobiose , Anaerobiose , Cromatóforos Bacterianos/metabolismo , Bacterioclorofilas/metabolismo , Carotenoides/metabolismo , Citocromos/metabolismo , Eletroforese em Gel de Poliacrilamida , Cinética , Luz , Rhodobacter sphaeroides/metabolismo , Espectrofotometria , Espectrofotometria InfravermelhoRESUMO
Site-directed mutagenesis has suggested that conserved histidine and alanine residues in the alpha-subunit of the B880 (LHI) antenna complex of Rhodobacter capsulatus (alpha His32 and alpha Ala28) form part of the bacteriochlorophyll binding site (Bylina, E.J., Robles, S.J. and Youvan, D.C. (1988) Isr. J. Chem. 28, 73-78). Spectroscopic characterization of chromatophores from alpha Ala28 mutants at 77 K revealed: (i) red shifts in B880 absorption and emission maxima of approximately 6 and 10 nm, respectively, with a serine exchange; (ii) red shifts of 3 nm with a glycine exchange; (iii) and no significant shifts with a cysteine exchange, despite a reduction of approximately 50% in B880 level. The strains with the serine and glycine exchanges showed characteristic fluorescence polarization increases over the red-edge of the B880 band, suggesting that the absorption red shifts arose from altered pigment-protein interactions rather than from increased oligomerization states that would be expected to show markedly diminished and red shifted rises in polarization (Westerhuis, W.H.J., Farchaus, J.W. and Niederman, R.A. (1993) Photochem. Photobiol. 58, 460-463). Excitation spectra of strains with alpha His32 to glutamine and alpha Ala28 to histidine exchanges, thought to be depleted in B880, revealed low levels of a 'pseudo-B880' complex with blue-shifted maxima and fluorescence polarization rises; when excited directly into this component, the former strain showed an emission spectrum similar to that of B880. An essentially wild-type electrochromic carotenoid response was observed only in the B880-containing mutants, since membranes isolated from the B880-depleted strains exhibited an increased permeability to ions.
Assuntos
Bacterioclorofilas/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter capsulatus/genética , Sequência de Aminoácidos , Bacterioclorofilas/química , Sítios de Ligação , Sequência Consenso , Polarização de Fluorescência , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/química , EspectrofotometriaRESUMO
Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. The material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll alpha and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.
Assuntos
Cromatóforos Bacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Bacterioclorofilas/biossíntese , Clorofila/análogos & derivados , Membranas Intracelulares/metabolismo , Rhodobacter sphaeroides/metabolismo , Membrana Celular/metabolismo , Cloranfenicol/farmacologia , Rhodobacter sphaeroides/efeitos dos fármacos , Tetraciclina/farmacologiaRESUMO
Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromotophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (Em = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.
Assuntos
Proteínas de Bactérias/análise , Bacterioclorofilas/análise , Clorofila/análogos & derivados , Rhodobacter sphaeroides/metabolismo , Cromatóforos Bacterianos/metabolismo , Escuridão , Membranas Intracelulares/metabolismo , Cinética , Luz , Proteínas de Membrana/análise , Oxirredução , Fotossíntese , EspectrofotometriaRESUMO
The complete amino acid sequence was determined for the alpha- and beta-chains of the B875 light-harvesting protein purified from photosynthetic membranes of Rhodopseudomonas sphaeroides 2.4.1. The sequence of the B875-alpha-polypeptide was identical to that reported for the R26.1 carotenoidless mutant [(1985) Biochim. Biophys. Acta 806, 185-186] and contained 58 amino acid residues with a blocked methionine and a glutamic acid at the N- and C-termini, respectively. The B875-beta-polypeptide contained 48 amino acid residues with alanine and phenylalanine as respective N- and C-termini; although otherwise identical, the leucine at position 29 in the wild-type strain was replaced by proline in the mutant. This radical amino acid substitution occurred within the central hydrophobic domain of the beta-polypeptide chain and is thought to result in a weakening of the structure of the alpha/beta heterodimer since it was not possible to isolate the intact pigment-protein complex from the R26.1 mutant strain.
Assuntos
Proteínas de Bactérias , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Carotenoides/deficiência , Carotenoides/genética , Complexos de Proteínas Captadores de Luz , Substâncias Macromoleculares , Mutação , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética , Rhodobacter sphaeroides/genética , Relação Estrutura-AtividadeRESUMO
The role of phospholipid in the structural organization of the light-harvesting complexes of Rhodopseudomonas sphaeroides was examined in photosynthetic (chromatophore) membrane vesicles fused with liposomes. Photochemically active preparations with progressive phospholipid enrichment up to greater than 15-fold were obtained by both polyethylene glycol- and acidic-pH-induced fusion. Their fluorescence emission at approximately 300 and 77 K was increased by 2-3.5-fold from the peripheral B800-850 antenna relative to that from the core B875 antenna. Up to 30-40% reduction in the efficiency of excitation energy transfer between B850 and B875 was also observed at 77 K suggesting a selective, phospholipid-induced dissociation of a portion of the B800-850 from the rest of the light-harvesting system.
Assuntos
Cromatóforos Bacterianos/metabolismo , Lipossomos/metabolismo , Rhodobacter sphaeroides/metabolismo , Centrifugação com Gradiente de Concentração , Transferência de Energia , Fosfolipídeos/metabolismo , Fotoquímica , Espectrometria de FluorescênciaRESUMO
Under carefully controlled ionic conditions, large-scale preparations of highly purified chromatophores and cell envelopes were obtained from phototrophically grown Rhodopseudomonas spheroides by zonal ultracentrifugation. The majority of the bacteriochlorophyll a was located in a single, discrete chromatophore band, whereas the envelopes were nearly devoid of photopigment. The envelope fraction contained substantial quantities of succinic dehydrogenase and cytochromes, confirming that phototrophically grown cells contain a photopigment-deficient cytoplasmic membrane. Magnesium at concentrations of 1.0 mM or higher caused chromatophores to reversibly aggregate with the cell envelope. Significant aggregation was also promoted by other divalent metals (Co(2+) > Mn(2+) > Ca(2+) > Mg(2+)), but aggregation was less extensive with monovalent cations. These results account for the distribution of photopigments in two bands reported by others and further suggest that the photosynthetic apparatus of R. spheroides is located on membranes largely distinct from the cell wall-cytoplasmic membrane complex.
Assuntos
Cromatóforos Bacterianos , Membrana Celular , Rodopseudomonas/citologia , Aerobiose , Cromatóforos Bacterianos/análise , Cromatóforos Bacterianos/enzimologia , Proteínas de Bactérias/análise , Carboidratos/análise , Membrana Celular/análise , Membrana Celular/enzimologia , Sistema Livre de Células , Centrifugação com Gradiente de Concentração , Centrifugação Zonal , Clorofila/análise , Citocromos/análise , Lipídeos/análise , Magnésio/farmacologia , Ácidos Nucleicos/análise , Fósforo/análise , Rodopseudomonas/análise , Rodopseudomonas/enzimologia , Espectrofotometria , Succinato Desidrogenase/metabolismo , TrítioRESUMO
Proteinase K was used to degrade membrane proteins exposed at the outer (cytoplasmic) and inner (periplasmic) surface of sealed, uniformly oriented chromatophore vesicles of Rhodobacter sphaeroides. Exclusive and controlled digestion of the chromatophore interior was achieved after Ca(2+)-induced fusion with large unilamellar phosphatidylglycerol liposomes containing microencapsulated enzyme. Reaction center subunit H, which served as a marker for the outer surface, was degraded to a slightly smaller product in chromatophores. This protein remained intact after liposome-chromatophore fusion, suggesting that the intermixing of lipid bilayers proceeded without significant leakage of the aqueous vesicle contents. In contrast, while cytochrome c1 was not affected in chromatophores, 70-75% was degraded within 60 min after liposome-chromatophore fusion. These results support an arrangement in which the bulk of this protein, including the mesoheme component and active site residues, faces the periplasmic side of the membrane. Although current functional models for the cytochrome bc1 complex predict that the Rieske iron-sulfur center interacts with cytochrome c1 in the periplasm, the iron-sulfur protein resisted proteolytic attack in the liposome-chromatophore fusion products under conditions that caused extensive degradation of cytochrome c1. Two cleavage products of the iron-sulfur protein were observed after the digestion of chromatophores, suggesting both a heterogeneity in the population of this protein and the exposure of at least part of its molecular mass to the cytoplasm.
Assuntos
Cromatóforos Bacterianos/química , Citocromos c1/análise , Complexo III da Cadeia de Transporte de Elétrons , Proteínas Ferro-Enxofre/análise , Rhodobacter sphaeroides/química , Cálcio/metabolismo , Membrana Celular/metabolismo , Endopeptidase K , Lipossomos , Fosfatidilgliceróis/metabolismo , Serina EndopeptidasesRESUMO
The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800-850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a 'free' pigment band; the remaining absorption appeared at 880 nm and shifted to â¼845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min(-1) for the initial rapid change and 0.05 and 0.11 min(-1) for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.
RESUMO
Vibrio succinogenes required relatively small amounts of succinate for growth when formate plus nitrate was supplied as the energy source. The requirement for succinate was not apparent when formate plus fumarate was the energy source because fumarate is reduced to succinate. l-Asparagine, fumarate, and malate replaced succinate, and it appears likely that they do so by being converted to succinate. Formate plus l-aspartate or l-asparagine served as energy sources for growth. The stoichiometry of the reduction of aspartate with H(2) by resting cells suggests an aspartase reaction followed by reduction of fumarate to succinate. Oxalacetate or pyruvate plus bicarbonate did not substitute for succinate, nor did many other compounds that were tested. (14)C-succinate was mainly incorporated into the alcohol-soluble fraction of cells, although there was significant incorporation into the hot trichloroacetic acid-soluble and -insoluble fractions.
Assuntos
Succinatos/metabolismo , Vibrio/metabolismo , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Isótopos de Carbono , Fracionamento Celular , Cromatografia , Meios de Cultura , Formiatos/metabolismo , Fumaratos/metabolismo , Malatos/metabolismo , Nitratos/metabolismo , Solventes , Espectrofotometria , Ácido Tricloroacético , Vibrio/crescimento & desenvolvimentoRESUMO
Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.
Assuntos
Cromatóforos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Membranas Intracelulares/metabolismo , Fotossíntese , Rhodobacter sphaeroides/metabolismo , Divisão Celular , Complexo de Proteínas do Centro de Reação Fotossintética , Rhodobacter sphaeroides/crescimento & desenvolvimento , Rhodobacter sphaeroides/ultraestruturaRESUMO
A protease delivery system was developed for the exclusive and controlled digestion of proteins exposed at the morphological inside (periplasmic surface) of Rhodobacter sphaeroides chromatophores. In this procedure, proteinase K is encapsulated within large unilamellar liposomes which are fused to the chromatophores in the presence of Ca2+ ions. The liposomes were prepared by a detergent dialysis procedure from native phosphatidylglycerol and found to undergo rapid bilayer fusion with purified chromatophore preparations above a threshold concentration of 12.5 mM CaCl2. The fusion process was complete within 10 min at 35 mM Ca2+ with about 80% of the pigment located in the fusion products. Electron micrographs of freeze-fracture replicas confirmed the intermixing of the lipid bilayers and the unilamellar structure of the fused membrane vesicles. The procedure did not affect the labile B800 chromophore of the B800-850 antenna complex, but reduced slightly the absorption due to the B875 core antenna. Emission from both light-harvesting complexes was increased in the fused membranes, suggesting a partial dissociation of photosynthetic units in the expanded bilayer. The results, together with those presented in the following paper (Theiler, R., and Niederman, R. A. (1991) J. Biol. Chem. 266, 23163-23168), demonstrate that this new method fulfills the stringent requirements for a successful delivery of macromolecules to the chromatophore interior.
Assuntos
Cromatóforos Bacterianos/química , Proteínas de Bactérias/análise , Cálcio/metabolismo , Proteínas de Membrana/análise , Fosfatidilgliceróis/metabolismo , Rhodobacter sphaeroides/química , Cromatóforos Bacterianos/ultraestrutura , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Transporte Biológico , Membrana Celular/metabolismo , Cromatografia em Camada Fina , Endopeptidases/metabolismo , Técnica de Fratura por Congelamento , Cinética , Lipossomos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Rhodobacter sphaeroides/metabolismoRESUMO
The ubiquinol-cytochrome c2 oxidoreductases (cytochrome bc1 complex) of Rhodobacter sphaeroides contains highly conserved cytochrome b, cytochrome c1 and Rieske FeS subunits, as well as a unique 14 kDa polypeptide, designated as subunit IV, thought to function as a ubiquinol-binding protein [Yu and Yu (1991) Biochemistry 30, 4934-4939]. As the topology of subunit IV is unknown and that of the FeS subunit remains a matter of debate, both the inner (cytoplasmic) and outer (periplasmic) surfaces of the intracytoplasmic membrane (ICM) were digested with proteinase K, and cleavage products were identified by immunoblotting. In uniformly oriented chromatophore vesicles (inner ICM surface exposed), fragments of approx. 4 and 1 kDa were removed from subunit IV and the FeS protein respectively. Neither subunit IV nor the FeS protein was cleaved from the outer ICM surface as exposed in osmotically protected spheroplasts or as presented to proteinase K after microencapsulation of the protease in unilamellar liposomes and fusion of these structures to chromatophore vesicles. Studies with the isolated bc1 complex, however, suggested that the C-terminal domain of the Rieske FeS, thought to reside on the periplasmic side of the ICM, was resistant to proteinase K. Overall, these results suggest a single N-terminal transmembrane helix for the FeS protein, with exposure of the N-terminus to the cytoplasm and an orientation in which a major, N-terminal portion of subunit IV is located in the cytoplasm with the predicted C-terminal transmembrane domain anchoring this polypeptide to the membrane.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/análise , Proteínas Ferro-Enxofre/análise , Rhodobacter sphaeroides/química , Cromatóforos Bacterianos/metabolismo , Membrana Celular/química , Quimotripsina/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Immunoblotting , Proteínas Ferro-Enxofre/metabolismo , Fragmentos de Peptídeos/metabolismo , Rhodobacter sphaeroides/ultraestrutura , Serina Endopeptidases/metabolismo , Esferoplastos/metabolismoRESUMO
Highly purified preparations of cytoplasmic and outer membrane were isolated from aerobically grown Rhodospirillum rubrum lysed by sequential treatment with lysozyme, ethylenediaminetetraacetate, and Brij 58. The membranes were resolved and separated from other cellular constitutents by a combination of velocity and isopyknic sedimentation in sucrose density gradients. On the basis of their appearance in electron micrographs and their protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these preparations appear to be quite similar to those obtained from other gram-negative bacteria. The cytoplasmic membrane fraction contained the majority of the total membrane-bound succinic dehydrogenase activity and was 10-fold enriched in b- and c-type cytochrome with respect to the outer membrane. The latter fraction was characterized by a much greater carbohydrate content and the presence of arachidic acid, which is typical of R. rubrum lipopolysaccharide. Their protein fatty acid, and overall chemical compositions suggested that these preparations were freer from cross-contamination than those obtained from R. rubrum with currently available methods.
Assuntos
Rhodospirillum rubrum/análise , Aerobiose , Proteínas de Bactérias/análise , Bacterioclorofilas/análise , Fracionamento Celular , Membrana Celular/análise , Parede Celular/análise , Citocromos/análise , Ácido Edético/farmacologia , Ácidos Graxos/análise , Peso Molecular , Muramidase/farmacologia , Peptídeos/análise , Fosfolipídeos/análise , Rhodospirillum rubrum/crescimento & desenvolvimento , Rhodospirillum rubrum/ultraestrutura , Tensoativos/farmacologiaRESUMO
Isolation of highly purified membrane fractions from phototrophically grown Rhodospirillum rubrum was achieved by velocity and isopyknic sedimentation under carefully controlled ionic conditions. Bacteriochlorophyll-rich and succinic dehydrogenase-rich chromatophores that were essentially devoid of contamination by non-chromatophore protein were separated from a denser fraction in extracts disrupted in a French pressure cell. Highly purified chromatophores and a nearly photopigment-free envelope fraction were also obtained from cells lysed by treatment with ethylenediaminetetraacetate-lysozyme-Brij 58. After lysis with lysozyme and ethylenediaminetetraacetate alone, about 50% of the total photosynthetic pigment was released in chromatophores similar to those isolated by the above procedures. Chromatophores prepared by each method were found to have very similar near-infrared absorption spectra, overall chemical composition, equilibrium buoyant densities in CsCl, and protein patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles of the dense, outer membrane-rich fractions were different from those of the chromatophores. The release of much of the photosynthetic apparatus as discrete chromatophores is osmotically lysed extracts necessitates a reevaluation of the concept that isolated chromatophores arise only from mechanical comminution of a larger membrane structure.
Assuntos
Rhodospirillum rubrum/análise , Cromatóforos Bacterianos/análise , Proteínas de Bactérias/análise , Bacterioclorofilas/análise , Carboidratos/análise , Fracionamento Celular , Citocromos/análise , Ácido Edético/farmacologia , Muramidase/farmacologia , Osmose , Fosfolipídeos/análise , Rhodospirillum rubrum/ultraestrutura , Tensoativos/farmacologiaRESUMO
The particulate hydrogenase of Vibrio succinogenes is solubilized during treatment of cell envelopes at pH 11.0. Alkali-solubilized enzyme requires sulfhydryl compounds for activity. At neutral pH, soluble enzyme is reincorporated into alkalitreated cell envelopes and no longer requires an additional activator. In the present study, cell envelopes prepared by lysing cells with ethylenediaminetetraacetic acid plus lysozyme (EDTA-lysozyme) were used to determine the chemical composition of cell envelopes and derived pH 11.0 soluble and insoluble fractions and to investigate some properties of the binding and activation of alkali-solubilized hydrogenase. Lysis with EDTA-lysozyme resulted in the formation of spheroplast ghosts. The derived cell envelopes contained 61% protein, 3% ash, 23% lipid, and 1% phosphorus. The alkali-treated cell envelopes contained 50% protein, 2% ash, 24% lipid, and 1% phosphorus. The ash from cell envelopes and alkali-treated cell envelopes was rich in iron and phosphorus and also contained calcium, copper, magnesium, sodium, and zinc. Virtually all of the weight of the ashed samples was accounted for by the oxides of these metals. Since the reconstitution of particulate hydrogenase was achieved with pH 11.0 supernatant solution and precipitate, intact mucopeptide is not essential for hydrogenase binding. Release of hydrogenase during EDTA-lysozyme lysis was found to depend upon an apparent structural change which occurs in the membranes during extended storage at -20 C.
Assuntos
Oxirredutases , Vibrio/enzimologia , Bacteriólise , Membrana Celular/análise , Membrana Celular/enzimologia , Parede Celular/análise , Parede Celular/enzimologia , Ácido Edético/farmacologia , Hidrogênio , Concentração de Íons de Hidrogênio , Muramidase/farmacologia , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Vibrio/análise , Vibrio/efeitos dos fármacosRESUMO
The pathway of arginine biosynthesis in Streptococcus bovis was studied by radioactive tracer techniques. Cells were grown anaerobically with (14)CO(2) in a synthetic medium containing NH(4) (+) as the sole nitrogen source except for the trace present in nitrogen-containing vitamins. The protein fraction isolated from the labeled cells was acid-hydrolyzed, and (14)C-arginine was isolated from the protein hydrolysate by ion-exchange chromatography. The carboxyl carbon of the isolated arginine was removed with arginine decarboxylase, and the guanidino carbon was removed by simultaneous arginase-urease degradation. By manometric measurement and liquid scintillation counting of the CO(2) released by enzymatic degradation, 50% of the label was found in the carboxyl carbon and 50% in the guanidino carbon. Specific radioactivity determinations indicated that growth on (14)CO(2) resulted in twice as much label in arginine as with aspartate, glutamate, or lysine. These results are consistent with a glutamate --> ornithine --> citrulline pathway of arginine biosynthesis in S. bovis and provide further evidence for the synthesis of glutamate via the tricarboxylic acid cycle reactions from citrate through alpha-ketoglutarate.