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Hydrogen metabolism in the facultative anoxygenic cyanobacteria (blue-green algae) Oscillatoria limnetica and Aphanothece halophytica.

Two facultative anoxygenic photoautotrophic cyanobacteria, Oscillatoria limnetica and Aphanothece halophytica were found capable of CO2 photoassimilation using molecular hydrogen as electron donor in a photosystem I driven reaction. A. halophytica was also capable of evolving hydrogen from Na-dithionite reduced methylviologen in a light independent reaction.

Anaerobiosis↗

p-Cresol formation by cell-free extracts of Clostridium difficile.

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O2-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresol-forming enzyme could not be purified.

Butyrates↗

The direct measurement of cytochrome P450 in unfixed tissue sections.

A method for the measurement of cytochrome P-450 in unfixed cryostat sections is described. The sections are incubated for 10 minutes at room temperature in a buffered solution containing polyvinyl alcohol and sodium dithionite. Two incubations are performed on serial sections, one in nitrogen and the other in carbon monoxide. Readings are taken on a Vickers M85 microdensitometer fitted with a high sensitivity photomultiplier amplifier system, the measurements being made on corresponding fields in the serial sections. Subtraction of the nitrogen values from the carbon monoxide values, after allowing for an absorption shift, gives the absolute spectrum of cytochrome P450. The subtraction corrects for the tissue content of other haem-containing proteins. The cytochrome P450 spectrum shows a sharp maximum at 450 nm, and two other minor components absorbing at 444 nm and 458 nm. The content of cytochrome P450 in animals fed with phenobarbitone was 2.4 times greater than in control animals.

Animals↗

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids, dromedary camel and goat.

To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O2 transfer, the kinetics of O2 uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37 degrees C with a stopped-flow technique. O2 transfer conductance of RBC (G) was estimated from the rate of O2 saturation change and the corresponding O2 pressure difference between medium and hemoglobin. For comparison, O2 kinetics for the RBC of a low-altitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O2 transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O2 release into dithionite solutions of sufficiently high concentration (greater than 30 mM). The G values measured in these 'standard' conditions, Gst (in mmol X min-1 X Torr-1 X (ml RBC)-1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O2 diffusivity which depends on the concentration of cellular hemoglobin. The high Gst of RBC of high-altitude camelids may be considered to enhance O2 transfer in lungs and tissues. But the O2 transfer conductance of blood, theta, equal to Gst multiplied by hematocrit (in mmol X min-1 X Torr-1 X (ml blood)-1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).

Altitude↗

Studies on iron uptake and micelle formation in ferritin and apoferritin.

Iron uptake and micelle formation in ferritin and apoferritin have been followed both spectrophotometrically and by means of sedimentation velocity experiments. Information was thus obtained on the molecular weight distribution of the reconstitution product. To achieve incorporation 'native' ferritin (whole ferritin as purified from horse spleen), 'native' apoferritin (apoferritin prepared by fractionation of ferritin preparations) and 'reduced' apoferritin (apoferritin prepared by reduction of ferritin by dithionite or ascorbic acid) have been incubated with ferrous salts in the presence of oxidizing agents under different experimental conditions. Although some iron is incorporated in 'native' ferritin, full saturation is not achieved and the molecular weight distribution of the incubated products remains heterogeneous. 'Native' and 'reduced' apoferritin show a similar iron incorporation, but the reconstitution products markedly differ in terms of their iron distribution. Ferritin reconstituted from 'native' apoferritin has a broad molecular weight distribution, while that reconstituted from 'reduced' apoferritin is characterized by a narrow, homogeneous molecular weight distribution. However treatment of apoferrition with reducing or oxidizing agents prior to the incubation alters the characteristics of the iron distribution without changing the iron incorporation properties. These results point to a role of the protein moiety not only in iron oxidation, but also in micelle formation.

Animals↗

Role of tyrosine residues in cytoplasmic aspartate aminotransferase from beef kidney.

Cytoplasmic aspartate aminotransferase from beef kidney loses 25% of its activity on nitration with tetranitromethane while the apoenzyme about 95%. In the holoenzyme 0.5 tyrosine residue and 1.0 tyrosine residue in the apoenzyme are nitrated per enzyme protomer. In addition 1 cysteine residue per protomer is oxidized in both. The presence of substrates, alpha-ketoglutarate and glutamate, both at ten times their Km values, does not change these results. Mercaptoethanol does not affect the residual activity of either the nitrated holo or apoenzyme. Dithionite abolishes the activity of the nitrated holoenzyme by reducing tha coenzyme moiety. It has no effect on the native holoenzyme or on either the native or nitroapoenzyme.

Animals↗

Inhibition of fading in fluorescence microscopy of fixed cells.

Following Hirschfeld et al., dithionite was added to mounting media to inhibit fluorescence fading. Excellent response is reported for fluorescein, acridine orange, 33258 Hoechst, acriflavine, berberine (and ethidium bromide), but not for quinacrine.

Acridine Orange↗

A major integral protein of the plant plasma membrane binds flavin.

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Amino Acid Sequence↗

High-pressure stopped-flow spectrometry at low temperatures.

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.

2,6-Dichloroindophenol↗

Biochemistry without oxygen.

Published procedures for experimentation under anoxic conditions generally involve specialized apparatus that hinders the easy manipulation of experimental samples. We describe here some procedures that rapidly remove oxygen from experimental solutions, maintain anoxia with simple equipment for long periods of time, and do not interfere with normal sample addition and removal, spectrometric measurements, chromatographic manipulations, and the like. Anoxia can be achieved and maintained by the use of an enzyme system (glucose oxidase, glucose, catalase), or an inorganic oxygen-reducing system (ferrous pyrophosphate), or dithionite. Physical isolation of experimental samples from atmospheric oxygen can be maintained by continuous flushing with treated argon gas and/or by an overlay of heavy mineral oil.

Argon↗

Stoichiometric characteristics and resonance Raman spectra of azidosemimethemerythrin.

With extra precautions to remove dissolved oxygen, reduction of aquomethemerythrin with dithionite occurs with a stoichiometry very close to 1.0. The aquosemimethemerythrin so produced combines with N3- ion almost on a 1:1 basis per monomer. Resonance Raman spectra of azidosemimethemerythrin differ from those of azidomethemerythrin in the position of the Fe-N stretching frequency and in the absence of a feature corresponding to a mu-oxo bridge, but in both complexes the internal azide stretch occurs at the same frequency and the substitution of isotopically unsymmetrical 15N14N-2 leads to a splitting of the Fe-N band. These observations provide some insight into the structural features of the binuclear functional site.

Animals↗

Variable fluorescence of photosystem I particles and its application to the study of the structure and function of photosystem I.

Chlorophyll a fluorescence in Photosystem I (PSI) particles isolated according to the method of Bengis and Nelson [J. Biol. Chem. 252, 4564-4569 (1977)] was found to be dependent on the redox state of both P700 and X (an acceptor on the reducing side of PSI). Addition of dithionite plus neutral red to PSI caused an increase in fluorescence intensity and a shift of the main fluorescence peak from 689 to 674 nm. Addition of electron acceptors such as ferredoxin and methyl viologen decreased the fluorescence yield when added to PSI incubated under anaerobic conditions in the presence of excess dichlorophenol indophenol (DCIPH2). The Km for ferredoxin agreed with that determined from direct measurements of ferredoxin reduction, showing that X is a quencher of fluorescence. P700 was also found to be a quencher of fluorescence, since electron donors such as DCIPH2, TMPD, and plastocyanin decreased fluorescence with Km's nearly identical to those observed for P700+ reduction. Chemical modification of PSI (with ethylene diamine + a water-soluble carbodiimide) to make it positively charged increased the fluorescence yield and shifted the 689-nm peak to 674 nm. The Km's for DCIPH2 and ferredoxin were decreased. In contrast, modification of PSI with succinic anhydride, which increased the net negative charge, increased the Km for ferredoxin. Salts affected the interaction of methyl viologen with PSI. Both anion and cation selectivity were observed. Limited proteolysis increased the Km for both methyl viologen and ferredoxin, indicating that their binding site on PSI was altered. These results suggest that the binding site for ferredoxin is on either the 70- or the 20-kDa subunit of PSI.

Chlorophyll↗

Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans.

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

Cell Membrane↗

Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties.

The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.

Acetylene↗

Cytochrome b-563 redox changes in intact CO-fixing spinach chloroplasts and in developing pea chloroplasts.

Intact spinach chloroplasts, capable of high rates of photochemical oxygen evolution with CO2 as electron acceptor (120-350 mumol O2 mg chlorophyll-1 h-1) were examined for cytochrome redox changes. The response of the cytochromes in intact chloroplasts to oxidants and reductants appears to be governed by the permeability of the chloroplast envelope. The low potential cytochromes (b-559LP and b-563) were more slowly reduced at 25 degrees C by dithionite than is the case with broken chloroplasts. At 0 degrees C, the reduction of the low potential cytochromes in intactchloroplasts was extremely slow. The chloroplast envelope is impermeable to ferricyanide, slowly permeable to ascorbate and rapidly permeable to reduced dichlorophenolindophenol. Light-induced redox changes of cytochrome b-563 in intact chloroplasts were examined both at 0 degrees and 25 degrees C. A red/far-red antagonism on the redox changes of cytochrome b-563 was observed at 0 degrees C under anaerobic conditions. 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea (DCMU) inhibited the photoreduction of cytochrome b-563 in red light following far-red illumination. The photooxidation of cytochrome b-563 under anaerobic conditions was not influenced by DCMU or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The photoreduction of cytochrome b-563 under aerobic conditions was much less efficient than its photooxidation under anaerobic conditions. Developing pea chloroplasts showed much greater light-induced redox changes of cytochrome b-563 than did intact spinach chloroplasts. Our data are consistent with the view that cytochrome b-563 functions on a cyclic pathway around Photosystem I, but it appears that cyclic flow is sensitive to the relative poising of the redox levels of cytochrome b-563 and the components of the non-cylic pathway.

Aging↗

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2 degrees K.

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2 degrees K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are D = 0.02794 +/- 0.00007 cm-1, E = 0.00382 +/- 0.00007 cm-1, i.e. equal to those of monomeric chlorophyll a to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperatures 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.

Chloroplasts↗

Evidence that the intermediate electron acceptor, A2, in photosystem I is a bound iron-sulfur protein.

Absorption changes accompanying the formation of light-induced P-700+ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P700+ and A-2. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 in equilibrium A2 were completely lost. These results provide evidence that the intermediate electron acceptor, A2, is a bound iron-sulfur protein. Additional studies in the 400--500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A2 is different from that of P-430.

Cytochromes↗

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils.

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

Cytochrome b Group↗