Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

In vitro effects of some anaesthetic drugs on lactoperoxidase enzyme activity

In vitro effects of ketamine and bupivacaine drugs on bovine lactoperoxidase (LPO; E.C. 1.11.1.7) enzyme activity were investigated. Lactoperoxidase was purified with Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skimmed bovine milk. R_z(A₄₁₂/A₂₈₀) value for the purified LPO was found to be 0.8. Inhibition or activation effects of the drugs on LPO enzyme were determined using 2,2¹-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a chromogenic substrate at pH = 6.0. The I₅₀ values of ketamine and bupivacaine were 0.29 mM and 0.155 mM, respectively and the K_i constants for ketamine and bupivacaine were 0.019 ± 0.031 and 0.015 ± 0.021 mM, respectively; they were non-competitive inhibitors.     

Effects of natural shade on soybean thylakoid membrane composition

The effect of natural shade on chloroplast thylakoid membrane activity and composition was examined for soybean (Glycine Max. cv. Young) grown under field conditions. Plots with high (10 plants m^–1 row) or low (1 plant m^–1 row) plant density were established. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP). At 92 DAP, tagged leaves were used as reference points to characterize canopy light environments and isolate thylakoid membranes. Light environments ranged from a photosynthetic photon flux density (PPFD) of 87% of full sun to a PPFD of 10% of full sun. The decline in PPFD was accompanied by an increase in the far-red/red (735 nm/645 nm) ratio from 0.9 to approximately six. The major effects of shade on chloroplast thylakoid membranes were a reduction in chloroplast coupling factor and a shift in light-harvesting capacity from Photosystem I to Photosystem II. Photosynthetic electron transport capacity was not affected by differences in PPFD, but was 20 to 30% higher in the 1 plant m^–1 row treatment. The plant density effect on electron transport was associated with differences in plastocyanin concentration, suggesting that plastocyanin is a limiting factor in soybean. Shade did not have a significant effect on the concentration of Photosystem II, Cyt b₆f, or Photosystem I complexes.Abbreviations CF₁ chloroplast coupling factor - DAP days after planting - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - FR/R far-red/red - PBS 10 mM sodium phosphate (pH 7.0), 150 mM NaCl - PPFD photosynthetic photon flux density - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl - TTBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.05% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20) The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Mechanism of Cu2+-induced elevation of [3H]cimetidine binding to membranes in rat brain

The mechanism of increasing effect of CuCl₂ on specific [³H]cimetidine binding was examined in brain membranes of rats. CuCl₂-Induced elevation of [³H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl₂ (5–50 mM) inhibited effect of CuCl₂, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl₂ (5–50 mM) did not. CuCl₂ (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl₂ in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl₂ mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu²⁺ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl₂ also caused a marked elevation in [³H]cimetidine binding, seeming to be due to formation of cimetidine-Pd²⁺ chelate complex. There were two types of [³H]cimetidine binding in the presence of 20 nM PdCl₂: high affinity binding with K_d = 0.7 ± 0.1 nM and low affinity binding with K_d = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu²⁺ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone.     

Effects of phosphate on the activity, stability and regulatory properties of phosphoenolpyruvate carboxylase from the C4 plant Cynodon dactylon

The extraction of phosphoenolpyruvate carboxylase, PEPC (EC 4.1.1.31) from leaves of Cynodon dactylon (L.) Pers. with phosphate buffer (pH 7.4, 105 mM) was advantageous in comparison to the usual extraction with Tris-HCl buffer (pH 7.4, 100 mM); a higher activity was obtained, which was most evident at low substrate (phosphoenolpyruvate) concentrations. The PEPC activity was stable under dilution or in storage for at least 48 h at room temperature. The effects of phosphate buffer were not due to inhibition of phosphatase(s) action during the extraction, since they were also observed when the phosphates were added after the extraction with Tris-HCl. The phosphate-extracted enzyme was less responsive to both L-malate inhibition and activation by glucose-6-phosphate. The effects of phosphates might be due to preferential exclusion from the enzymic protein domain and, therefore, to a confinement of the enzyme to a fraction of the total volume. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of egg PC/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine

Context: Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required.

Objective: This study therefore aimed to provide extended RVC release and increased upload using modified liposomes.

Materials and methods: Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07?mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5?+?(NH₄)₂SO₄ and pH 7.4?+?(NH₄)₂SO₄) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4).

Results and discussion: An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5_in) or pH 7.4 with 250?mM (NH₄)₂SO₄ in the inner aqueous core (LMVV 7.4_in?+?ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ～70%) and sustained release (～25?h).

Conclusion: The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.     

A new procedure for fast isolation and purification of plastocyanin from the cyanobacterium Anabaena variabilis

Methods are described for growing the cyanobacterium A. variabilis and for the isolation and purification of plastocyanin from the grown culture. Cell paste which had been stored at –35°C was suspended in 1 mM MES buffer, pH 6.5 and centrifuged. The supernatant was diluted to a conductivity of 0.12 mS, [Fe(CN)₆]^3- added to a concentration of 0.5 mM and the solution loaded on a S Sepharose Fast Flow column. After elution and ultrafiltration, the plastocyanin containing fractions were reloaded on a S Sepharose Fast Flow column for final purification. A typical yield in three days from cells harvested from 3×20 l of medium was 32 mg plastocyanin with a minimum absorbance ratio A₂₇₈/A₅₉₇=1.14. This procedure is faster and the yield higher than for previous procedures.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - PC plastocyanin     

Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells

Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO₃)₂, 60 mM MgCl₂ and 30 mM NaHCO₃. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).     

Single-step chromatography for simultaneous purification of C-phycocyanin and allophycocyanin with high purity and recovery from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The cyanobacterium Spirulina (Arthrospira) platensis is a good source of phycobiliprotein purification. C-phycocyanin (C-PC) is the major phycobiliprotein, while allophycocyanin (APC) is less abundant in S. platensis. Previously reported methods for C-PC purification are only able to offer either high purity or high efficiency. This paper describes one-step anion exchange chromatography method with continuous pH gradient elution for simultaneous purification of C-PC and APC with high purity and high recovery. Crude C-PC and APC were extracted and concentrated by ammonium sulfate fractionation at saturation of 25% and 60%, then purified on a DEAE-Sepharose Fast Flow chromatography column with continuous pH gradient elution from pH 5.0 to 3.6. After this single-step chromatography, C-PC and APC with high purity and recovery were simultaneously obtained. The purity ratios of C-PC and APC reached 5.59 (A₆₂₀/A₂₈₀) and 5.19 (A₆₅₀/A₂₈₀), respectively. Their purity was further demonstrated by electrophoresis and fluorescence emission spectroscopy. Moreover, the total recovery yield of pure C-PC and APC were 67.04% and 80.0%, representing 111.83 and 29.28 mg·g⁻¹ lyophilized weight, respectively. The obtained C-PC and APC remained stable over a pH range of 4–9. This purification method for high purity and recovery of C-PC and APC proved to be fairly efficient compared with previously reported methods.     

Purification and some particle properties of anthriscus yellows virus, a phloem-limited semi-persistent aphid-borne virus

Anthriscus yellows virus (AYV), a phloem-limited virus transmitted in the semi-persistent manner by the aphid Cavariella aegopodii, was purified by treatment of leaf extracts with cellulasc, followed by differential and sucrose density gradient centrifugation. ‘The preparations contained isometric particles c. 29 nm in diameter which were unstable unless stored in buffer at pH 8.0 containing 1 mM CaCl₂,. The particles sedimented as two components, ’full‘ nucleoprotein particles with A₂₆₀/A₂₈₀= 1.83 containing about 42% nucleic acid, and ’empty‘ protein shells with A₂₆₀,/A₂₈₀= 0.73; their buoyant densities in CsCl solutions were 1.52 and 1.27 g/cm³. Respectively. Yields of ihe nircleoprotein particles were c. 1.75 mg/kg leaf tissue. The particles contained a single species of RNA, of mol. wt 3.6 × 10 “(10 000 nucleotides). Particle protein preparations contained four electrophoretic species, of mol. wt (× 10³) 35.0, 28.3, 23.3 and 22.3.C. aegopodii did not transmit AYV from purified preparations. A rabbit injected with AYV preparations produced antibodies that coated AYV particles in electron microscope tests, but gave variable reactions in immunosorbent electron microscopy (ISEM), depending on the composition of the medium. No reactions were obtained in enzyme-linked inimunosorbent asjay (ELISA). No serological relationship was detected in ISEM between AYV and any of 10 viruses that resembled it in one or more properties.     

Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purification,characterization and sulfur metabolism

Three cytochromes of the thiosulfate-utilizing green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum were highly purified by ion exchange column chromatography and ammonium sulfate fractionation. All three cytochromes are located in the soluble fraction. Cytochrome c-551 (highest purity index obtained: A₂₈₀/A₄₁₆=0.39) shows maxima at 551 nm (-band), 521 nm (-band), and 416 nm (-band) for the reduced form. This cytochrome is an acidic protein with a molecular weight of 32,000, a redox potential of 150 mV, and an isoelectric point at pH 6.0. Cytochrome c-553 (highest purity index obtained: A₂₈₀/A₄₁₇=0.8) is also an acidic protein with maxima at 553,5 nm, 523,5 nm and 417 nm for the reduced form, a molecular weight of 63,000, a redox potential of 90 mV, an isoelectric point at pH 6.3, and it contains FAD as flavin component. It is autoxidizable and participates in sulfide oxidation, but cannot catalyze the reverse reaction. The cytochrome c-555 (highest purity index obtained: A₂₈₀/A₄₁₈=0.16) is a small basic protein with maxima at 555 nm, 523 nm and 418 nm (reduced form), a molecular weight of 12,500, an isoelectric point between pH 10 and 10.5, and a redox potential of 155 mV. The ratio of the cytochrome contents to each other is constant and does not change when the organism has only thiosulfate or sulfide as the main electron donor in the medium.The soluble fraction further contains the non-heme ironcontaining proteins rubredoxin and ferredoxin. The anaerobic sulfide oxidation in a growing culture of Chlorobium vibrioforme f. thiosulfatophilum is accompanied by a rapid formation of thiosulfate, which is only utilized when sulfide is no longer available, while the elemental sulfur concentration increases constantly until thiosulfate is consumed.Non-common abbreviations C Chlorobium - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein     

Purification, N-terminal sequencing and partial characterization of a novel aspartic proteinase from the leaves of Medicago sativa L. (alfalfa)

A novel aspartic proteinase (EC 3.4.23) from Medicago sativa L. (alfalfa) was purified to homogeneity using Source Q ion-exchange, concanavalin-A Sepharose and pepstatin-A agarose affinity chromatography. The enzyme, M _r=33.5 kDa, is monomeric and catalyzes the cleavage of a broad spectrum of peptide bonds of hydrophobic amino acids from pH 2.6 to 6.4. The enzyme is inhibited by pepstatin-A and is consistent with the properties of an aspartic proteinase. The N-terminal amino acid sequence of the protein shows 50 and 40% similarity with the cyprosin and barley aspartic proteinases, respectively.     

A novel method for measuring human hepatic lipase activity in postheparin plasma

The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl(2), 1.5 mM CaCl(2), monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 microl of R-1 was incubated at 37 degrees C with 3 microl of samples for 5 min, and 80 microl of R-2 was added. Within-run coefficient of variations was 0.9-1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6-99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [(14)C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer.     

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.     

Use of phospholipase A to compare phospholipid organization in synaptic membranes,myelin, and liposomes

Summary The pattern of fatty acid release from rat synaptic membranes in the presence of phospholipase A₂ (Vipera russelli) was compared to that from liposomes comprised of phospholipids. Phospholipase A₂ more readily attacked myelin and synaptic membranes than liposomes prepared from total phospholipids derived from myelin. Although hydrolysis of liposomal phospholipids occurred in the absence of added calcium, the presence of 2mm CaCl₂ or 2% bovine serum albumin significantly enhanced the phospholipase attack of liposomes, but not synaptic membranes or myelin. Phospholipase exhibited a marked preference for phospholipids containing docosahexaenoic acid (226) in the synaptic membranes, while with liposomes the pattern of released fatty acid reflected the fatty acid composition in the two-position of the phospholipids. Although either calcium or albumin markedly increased the phospholipase hydrolysis of liposomes, neither affected the hydrolysis of synaptic membranes or the pattern of fatty acid release from liposomes. It was concluded that the nonlipid constituents, particularly the proteins, of biomembranes were responsible for the organization of the phospholipids and accounted for the observed differences between liposomes and synaptic membranes with respect to enzymic accessibility.     

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κ_m values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κ_i value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κ_i value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.     

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo ³¹P and ¹³C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H⁺. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [¹³C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [¹³C]ethanol was added.Abbreviations P_i inorganic phosphate - P_ic inorganic phosphate in the cytoplasm - P_iv inorganic phosphate in the vacuole - tP terminal phosphate in polyphosphate     

Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic ciliates using 16S rRNA binding oligonucleotide probes

The identity of ectosymbiotic bacteria of some marine, free-living anacrobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.Abbreviations DMSO dimethyl sulfoxide - PBS phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH: 7.3 - TEAA triethylamonium acetate - 1 x SSC standard sodium citrate buffer, 150 mM NaCl, 15 mM Na₂ citrate, pH: 7.0 - 1 x Denh Denhardts solution, 0.02% ficoll, 0.02% bovine serum albumine, 0.02% polyvinol-pyralidone     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.