Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

Temperature dependency of the Mg-ATPase and photophosphorylation activities in Rhodospirillum rubrum chromatophores

Arrhenius plots for ATP synthesis, coupled to endogenous and Phenazine methosulfate or N,N,N,N,-Tetramenthyl-1,4-Phenylene diamine-mediated photosynthetic election transport and for ATP hydrolysis were studied in Rhodospirillum rubrum chromatophores.Coupled or uncoupler induced Mg-ATPase show no discontinuity in the range tested (30°C-5°C) and they also have the same activation energy. Phenazine methosulfatecatalyzed photophosphorylation has also a single activation energy where as the endogenous reaction shows complex and ageing dependent behaviour, alternating temperature ranges having high (45.2 to 144,4 kJ·mol^-1) and very low (ca 0.0 to 3.3 kJ·mol^-1) activation energy.Abbreviations Bchl Bacteriochlorophyll - Ea Activation energy - FCCP Carbonyl Cyanide p. Trifluoromethoxy henyl Hydrazone - PMS phenazine methosulfate - TMPD N,N,N,N-Tetramethyl-1,4-Phenylene diamine - R Rhodospirillum     

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per m². Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.Isolated reaction centers are easily incorporated into liposomes. In freeze fractures of liposomes particles similar in shape and size, although less dense as in chromatophores are observed. In negative staining much smaller units of only 5 nm in diameter are found indicating that reaction centers occur in the membrane as tri- or tetramers. There is a strong correlation between particle density in chromatophores and titratable reaction centers remaining in these membranes after extraction of reaction centers by detergents; both values are in good agreement with the yield of reaction centers at a given detergent concentration.Abbreviations LDAO Lauryldimethylamine oxide - PF protoplasmic fracture face - EF exoplasmic fracture face     

Structural changes accompanying the irreversible oxidation of the chromatophore membrane from Rhodospirillum rubrum G9

The structure of the chromatophore membrane of the carotenoid-free mutant Rhodospirillum rubrum G9 and the effect of irreversible photooxidation upon this structure have been investigated using several physical techniques. Native chromatophore membranes undergo endothermic transitions in two temperature regions; at temperatures of about 0°C a broad reversible transition and between approx. 50 and 90°C several endothermic transitions are observed which are irreversible. The first transition can be assigned to the gel-to-liquid crystal transition of the lipid bilayer present in chromatophores; the irreversible one is attributed to changes mainly in the quarternary and possibily tertiary structure of membrane proteins. CD measurements showed that heating of chromatophores up to 70°C has no effect upon the protein secondary structure. Photooxidation has little effect on the structure and dynamics of the lipid bilayer in the chromatophore membrane. The order (or average conformation) of both the lipid polar groups and the hydrocarbon chains is hardly changed. However, the lipid phase transition is dramatically broadened and the protein-associated endothermic transitions are greatly reduced. This indicates that the major effect of photooxidation is upon lipid-protein and protein-protein interactions. Electron microscopy studies support this interpretation. It can be shown that the dense and regular packing of protein particles observed in the chromatophore membrane is lost as an effect of photooxidation. Instead, randomly distributed particles of varying size and shape are seen. These results are interpreted to mean that pigment-protein interactions are responsible for maintaining the native long-range order in the chromatophore membrane of R. rubrum G9. Destruction of the pigments by photooxidation leads to irreversible protein dissociation which in turn is followed probably by random protein reaggregation.     

Manufacture of liposomes by isopropanol injection: characterization of the method

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes. Particle size can be controlled by the variation of process parameters, such as stirring speed of the aqueous phase and injection flow rate of lipid-isopropanol solution. Diameter of vesicles obtained by this method is less affected by the nature of phospholipid, as well as lipid concentration, than in the ethanol-injection process. In addition, the vesicles are generally smaller (approximately 40–210?nm). Accurate characterization of the particles, by fluorescence, ³¹P-NMR, and cryo–transmission electron microscopy, showed that particles are formed of a single lipid bilayer around an aqueous cavity. We thus provide the scientific community with a fully characterized alternative method to produce unilamellar vesicles.     

A comparison of electron transport and photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum

The photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum chromatophores have been compared in respect to the effects of artificial electron carries [N-methyl-phenazonium methosulfate (PMS) and diaminodurene], reducing agents (ascorbate in particular), and various quinones in the absence and presence of the electron transport inhibitors antimycin A and dibromothymoquinone (DBMIB). In addition, the effects of both inhibitors on photosynthetic electron transport through cytochromes b and c has been followed. From the results obtained, it appears that in both organisms: a) ubiquinone functions as an electron carrier between the cytochromes, and b) both antimycin A and DBMIB inhibit cyclic electron flow in the segment ... cytochrome bubiquinone»cytochrome c ..., but at different sites. The systems apparently differ mainly in respect to the nature of the electron flow by-pass shunt that is evoked in the presence of PMS; thus, in R. rubrum, PMS catalyzes a shunt that by-passes both cytochrome b and ubiquinone, whereas in Rps. capsulata the PMS shunt seems to circumvent only ubiquinone.Abbreviations BChl bacteriochlorophyll - DAD diaminodurene=2,3,5,6-tetramethyl-p-phenylenediamine - DBMIB dibromothymoquinone=2,5-dibromo-6-isopropyl-3-methylbenzoquinone - HOQNO heptylhydroxyquinoline-N-oxide - PMS N-methylphenazonium methosulfate     

Occurrence of oscillations in ATP synthesis and hydrolysis in chromatophores of Rhodospirillum rubrum

The conditions under which an oscillatory behaviour is observed during net hydrolysis or synthesis of ATP in chromatophores of Rhodospirillum rubrum FR1 are described. In the case of ATPase the oscillations are observed at low temperature (ca. 11°C) in the dark after an initial transient behaviour. These oscillations are attenuated or disappear by the addition of an uncoupler.Oscillations are also observed during ATP synthesis. At 3°C the oscillations appear spontaneously if photophosphorylation is measured during a sufficiently long time. At 30°C the mere intercalation of a dark period also at 30°C is sufficient to trigger the oscillations in the following light period.Abbreviations Bchl Bacteriochlorophyll - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - PMS phenazine methosulfate - TMPD, N,N,N,N tetramethyl-1,4-phenylenediamine Dedicated to Prof. Dr. Gerhart Drews as a homage for his permanent example as hard worker and careful scientist and also for his remarkable human quality     

Complementation of a pleiotropic Nif-Gln regulatory mutant of Rhodospirillum rubrum by a previously unrecognized Azotobacter vinelandii regulatory locus

A spontaneous pleiotropic Nif^- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171:1017–1027]. In addition to being unable to grow on N₂ as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH _inf4 ^sup+ , (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH _inf4 ^sup+ up-shift, and (f) an inability to modify (³²P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.     

Circular dichroism spectra of oriented photoreaction center from Rhodospirillum rubrum

The circular dichroism spectra of oriented and unoriented photoreaction centers of Rhodospirillum rubrum are compared. Orientation is achieved by pressing photoreaction center suspended in polyacrylamide gel. The biphasic bands at 870 and 810 nm and at 630 and 600 nm undergo a rotatory strength decrease when measured in the direction of the pressure, but not when measured in the direction normal to the pressure. Such a decrease in oriented photoreaction center is consistent with the model according to which these bands are dimer exciton bands of the special pair bacteriochlorophyll.     

On the significance of electron transport systems for growth of Rhodospirillum rubrum

The inhibitory effects of 2-hydroxybiphenyl on various electron transport reactions of isolated membranes and growth in the presence of malate of either phototrophic or chemotrophic cells of Rhodospirillum rubrum were studied. 50% inhibition of both oxygen uptake of whole cells and growth under chemotrophic conditions (i.e. aerobiosis in the dark) was achieved in the presence of 0.09 mM 2-hydroxybiphenyl. With isolated membranes the same effect on NADH oxidase was obtained with 0.08 mM of inhibitor. Succinate dependent respiratory reactions were inhibited by 50% at a concentration of 0.36 mM. Growth under phototrophic conditions (i.e. anaerobiosis in the light) was inhibited by 50% in the presence of 0.17 mM (wild type strain) or 0.21 mM (blue-green mutant, strain VI) of 2-hydroxybiphenyl. Photophosphorylation and light dependent NAD⁺ reduction by succinate were inhibited by 50% at concentrations of 0.21 mM and 0.03 mM of inhibitor, respectively. After phototrophic growth of the organisms for about five doublings of cell mass in the presence of 0.18 mM of 2-hydroxybiphenyl coloured carotenoids could no longer be detected. Membrane fractions of such cultures exhibited normal activities of succinate cytochrome c reductase but activities of NADH cytochrome c reductase were decreased by 80%. In comparison with a blue green mutant, strain VI, of R. rubrum light induced absorbance changes at 865 nm as well as activities of photophosphorylation were unaffected. However, no activity of light dependent NAD⁺ reduction with succinate could be detected. The data indicate that cellular respiration as well as chemotrophic growth depend largely on NADH dependent respiration. Phototrophic growth, on the other hand, is limited by photophosphorylation while energy dependent reversed electron flow to NAD⁺, if at all, is of rathe minor importance.Abbreviation BChl bacteriochlorophyll     

P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Studies on the compartmentation of DOG metabolism in the brain

Using 31P-NMR studies we have observed that 1. 2-Deoxyglucose leads into the brain in vivo and in superfused cortical slices in vitro to a maximum concentration at between 45 and 60 min, when 80% of the material is in the phosphorylated form. 2. The phosphorylated DOG6P disappears from the n.m.r. spectra with a half-life of ca 130 min. 3. Two resonances of DOG6P are observed in the actively metabolising tissue, whereas only one is visible in deproteinised tissue extracts. This suggests that the DOG6P is in two separate compartments which differ in pH. 4. Compartmentation between mitochondria, nerve endings and cytoplasm was concluded to be unlikely from subcellular fractionation studies, but the possibility of compartmentation between neurones and glia could not be so clearly assessed.     

A P-NMR study of structural and functional aspects of phosphate and phosphonate distribution in Tetrahymena

³¹P-NMR has been used to study the chemical nature of cytoplasmic components of live Tetrahymena in a non-invasive manner. The technique has further been used to characterize the physical behaviour of lipids extracted from this organism. In particular, we have shown the presence of large quantities of pyrophosphate and of tripolyphosphate in acid extracts of the organism. These are not detectable in the live cell due to the motionally rigid nature of the storage granules. We have characterized the distribution of phosphonic acids in the organism and followed the phase behaviour of the extracted cell lipids. Aqueous dispersions of extracted lipid show both bilayer and non-bilayer behaviour in the range of the growth temperature. The phosphonolipid in Tetrahymena appears to play a role similar to that of phosphatidylethanolamine in regulating the phase behaviour of the membrane. The high degree of unsaturation in the fatty acids of Tetrahymena is most likely responsible for the polymorphic phase behaviour observed near the growth temperature.     

The lipopolysaccharides of Rhodospirillum rubrum,Rhodospirillum molischianum,and Rhodopila globiformis

The cell wall lipopolysaccharides from three phototrophic species of the alpha₁-group of Proteobacteria, Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis were isolated and chemically characterized. Sodium deoxycholate polyacrylamide gel electrophoresis patterns revealed that the lipopolysaccharides of all three species possess O-chains. They are composed of repeating units only in R. molischianum and R. globiformis. The presence of l-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate indicated core structures in all three lipopolysaccharides. Glucosamine was found as backbone amino sugar in lipid A of R. molischianum and R. rubrum, while R. globiformis has 2,3-diaminoglucose as backbone amino sugar. The latter species also differed from the two former ones in its content of hydroxy fatty acids (3-OH-14:0, 3-OH-16:0 in R. rubrum and R. molischianum and 3-OH-14:0, 3-OH-18:0 and 3-OH-19:0 (possibly iso- or anteisobranched) in R. globiformis).Abbreviations DOC-PAGE sodium deoxycholate polyacrylamide gel electrophoresis - GC/MS combined gas-liquid chromatography/mass spectrometry - KDO 2-keto-3-deoxyoctonate     

NMR studies of malaria P nuclear magnetic resonance of blood from mice infected with Plasmodium berghei

High resolution ³¹P-NMR has been used for the non-invasive observation of metabolites and metabolic rates in blood of normal mice and of mice infected with Plasmodium berghei, the causative agent of malaria. ³¹P-NMR was used to quantitate levels of 2,3-diphosphoglycerate in whole cells as a function of the degree of parasitemia and yielded good agreement with the results of enzymatic assays. The time-dependence of ³¹P metabolites was monitored in both normal and infected erythrocytes, greater rates of decay of 2,3-diphosphoglycerate being observed in malarial blood which correlate with the level of parasitemia. Very high metabolic rates of infected cells render measurement of intracellular pH unreliable on freshly drawn whole blood. When appropriate measures are taken to avoid this complication, no difference is observed in the intracellular pH of parasitized and non-parasitized erythrocytes from infected animals. In both normal and parasitized mice the intraerythrocytic pH is more acidic than that of the suspending medium by 0.15 pH unit at 25°C. Unlike free-living protozoa, the parasitic protozoan Plasmodium does not contain detectable levels of phosphonates or polyphosphates, in either whole cells or perchloric acid extracts thereof.     

The reconstitution of photophosphorylation and the effect of inorganic phosphate at different pH's on the phenazine methosulfate photoinhibition of Rhodospirillum rubrum chromatophores

Preillumination of R. rubrum membranes in the presence of 50M phenazine methosulfate produces an inhibition of their photophosphorylating capacity.At low pH's phosphate protects against this photo-inhibition, whereas increasing the pH eliminates this protective effect. The inhibition can be produced not only by preillumination at high pH in the presence of phenazine methosulfate, but also by preillumination at low pH (5.0) and then shifting the pH to 8.0 in the dark. We have also measured the effect of preillumination in the presence of an uncoupler such as carbonylcyanide p-trifluoromethoxyphenylhydrazone, and found that at normal pH's while photophosphorylation was protected, the uncoupler activated ATPase was inhibited pointing to a clear difference for both reactions and perhaps different structural requirements for their activities.The purified coupling factor protein isolated from either normal or photoinactivated membrane will reconstitute normal photophosphorylation in previously uncoupled membranes but not in uncoupled membranes which were inactivated by preillumination with phenazine methosulfate prior to uncoupling.     

Cation permeability induced by valinomycin,gramicidin D and amphotericin B in large lipidic unilamellar vesicles studied by 31P-NMR

Permeability induced by mobile carriers and channel-forming compounds in large unilamellar lipidic vesicles (LUV) has been studied by the proteon-cation exchange method. Proton movement has been monitored by pH-stat and ³¹P-NMR techniques. pH-stat measurements indicate that, in the presence of valinomycin, the proton efflux develops with a rate dependent upon valinomycin concentration, until equilibrium is reached. ³¹P-NMR spectra, monitoring pH-dependent intravesicular phosphate ionization, show that after addition of valinomycin the initial pH peak (pH 5.5; =0.25) shifts progressively to the position corresponding to the pH at equilibrium (pH 7.4; =2.20).In the presence of the channel-forming compounds, gramicidin D or amphotericin B, permeability developed in a few minutes whatever the concentration used. The percentage of total titratable proton released depends upon the antibiotic concentration. ³¹P-NMR spectra shows two signals from internal phosphate: one signal corresponding to the initial pH and a second signal corresponding to the pH at equilibrium indicating an all-or-none mode of action; just after addition of the antibiotic, two populations of vesicles coexist in proportions that depend on ionophore concentration; after longer incubation times all vesicles are permeabilized.The results obtained primarily reflect the differences in the mode of interaction with the membrane, of valinomycin as compared to the channel-forming reagents, gramicidin D or amphotericin B.     

Unidirectional inhibition of phosphoenolpyruvate carboxykinase from Rhodospirillum rubrum by ATP

The kinetic and regulatory properties of partially purified phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32) from Rhodospirillum rubrum were studied. The enzyme was active with guanosine-and inosinephosphates and must thus be classified as GTP (ITP): oxaloacetate carboxylyase (transphosphorylating). In the direction of oxaloacetate-formation, the enzyme was strongly inhibited by ATP (K_i=0.03 mM). ITP, UTP, CTP and GTP were less inhibitory. The inhibition was competitive with respect to GDP or IDP, but not with respect to PEP. In the direction of PEP-synthesis, the enzyme was not inhibited, but rather activated by ATP.     

Effect of lyophilization on liposomal encapsulation of salmon calcitonin

Purpose: The intent of this work was to assess the impact of lyophilization on the encapsulation of salmon calcitonin (sCT) into liposomes.

Methods: Four different liposomal formulations were investigated, i.e. DPPC:Chol:DSPE-PEG₂₀₀₀ (75:20:5 and 65:30:5) and DPPC:Chol (80:20 and 66.7:33.3). Lipid films were prepared and hydrated with loading buffer containing sCT and different concentrations of the cryoprotectant, trehalose dihydrate. The liposomes were lyophilized, reconstituted and extruded to obtain small unilamellar vesicles. Non-encapsulated sCT was separated by gel filtration. Non-lyophilized formulations and liposomes lyophilized without the cryoprotectant were used as controls. Liposomes were analyzed for particle size, polydispersity index, zeta-potential and encapsulation efficiency. ³¹P-NMR (phosphorous nuclear magnetic resonance spectroscopy) was performed on selected formulations.

Results: Post-lyophilization, no significant change in particle sizes and zeta-potentials were noted, regardless of the presence or absence of the cryoprotectant. Encapsulation efficiencies, however, increased following lyophilization, in both PEGylated (lyophilization control batch) and non-PEGylated liposomes (cryoprotectant batches only). ³¹P-NMR revealed the presence of two distinct vesicle populations – liposomes and micelles – in PEGylated formulation. The presence of micelles might be responsible for the observed encapsulation enhancement of sCT in the PEGylated formulation.

Conclusions: Lyophilization resulted in an increase in encapsulation efficiency of sCT in PEGylated liposomes, even in the absence of a cryoprotectant, due to presence of micellar vesicles.