Reversibility of oxygen switch-off effect on Bacillus polymyxa nitrogenase

1991 ◽  
Vol 37 (10) ◽  
pp. 775-779 ◽  
Author(s):  
Hans-Paul Holzmann ◽  
Heinrich A. Berghammer ◽  
Martin Ortner ◽  
Kurt Haselwandter
Keyword(s):  
Oxygen Consumption ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Cellular Respiration ◽  
Logarithmic Phase ◽  
Respiratory Protection ◽  
Bacillus Polymyxa ◽  
Closed Chamber ◽  
Oxygen Inhibition

The objective of this study was to analyse in vivo the effect of oxygen on the nitrogenase of Bacillus polymyxa. The culture technique employed in this study prevented spore formation by B. polymyxa during the entire period of exposure to acetylene. Under these conditions the acetylene-reduction assay allowed quantification of nitrogenase activity over long incubation periods (44 h). Nitrogenase activity was highest in cells harvested in the late logarithmic phase. At [Formula: see text] of 0.19 and 0.37 kPa, acetylene reduction was inhibited by 80 and 100%, respectively. This switch-off effect could be reversed through oxygen exhaustion, either by flushing the culture with N2 or by cellular respiration, suggesting a respiratory protection mechanism for the nitrogenase complex in B. polymyxa. Oxygen consumption measured by a closed-chamber respirometer showed a linear increase up to a [Formula: see text] of 0.2 kPa. Above 0.3 kPa a saturation in oxygen consumption was observed. Exposure to high oxygen pressures resulted in an irreversible loss of nitrogenase activity. The oxygen inhibition pattern was shown to be similar to that in other microaerophilic and anaerobic nitrogen-fixing microorganisms. Key words: Bacillus polymyxa, nitrogenase, oxygen inhibition, reversibility, respiration.

Hydrogen Evolution Catalyzed by Hydrogenase in Cultures of Cyanobacteria

1981 ◽  
Vol 36 (1-2) ◽  
pp. 87-92 ◽  
Author(s):  
Patrick C. Hallenbeck ◽  
Leon V. Kochian ◽  
John R. Benemann
Keyword(s):  
Hydrogen Production ◽  
Hydrogen Evolution ◽  
High Frequency ◽  
Nitrogenase Activity ◽  
Inhibitory Effect ◽  
Hydrogenase Activity ◽  
Oxygen Inhibition ◽  
Oxygen Sensitive

Abstract Cultures of Anabaena cylindrica, grown on media containing 5 mᴍ NH4Cl (which represses heterocyst formation), evolved hydrogen after a period of dark incubation under an argon atmosphere. This hydrogen production was not due to nitrogenase activity, which was nearly undetectable, but was due to a hydrogenase. Cultures grown on media with tungsten substituted for molybdenum had a high frequency of heterocysts (15%) and inactive nitrogenase after nitrogen starvation. The hydrogenase activity of these cultures was three-fold greater than the activity of non-heterocystous cultures. The effects of oxygen inhibition on hydrogen evolution by hetero-cystous cultures suggest that two pools of hydrogenase activity exist - an oxygen sensitive hydrogen evolution in vegetative cells and a relatively oxygen-resistent hydrogen evolution in heterocysts. In either case, inhibition by oxygen was reversible. Light had an inhibitory effect on net hydrogen evolution. Hydrogen production in vitro was much higher than in vivo, indicating that in vivo hydrogenase activity is limited by endogenous reductant supply.

Photorespiration and nitrogenase activity in the blue-green alga, Anabaena cylindrica

1972 ◽  
Vol 180 (1058) ◽  
pp. 87-102 ◽  
Keyword(s):  
Green Alga ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Dark Respiration ◽  
Reducing Power ◽  
Blue Green Alga ◽  
Anabaena Cylindrica ◽  
Oxygen Inhibition ◽  
Highly Sensitive ◽  
Photosynthetic Oxygen

Oxygen uptake in the light (photorespiration) by the nitrogen-fixing blue-green alga Anabaena cylindrica may be up to twenty times the dark respiration rate. The rate of uptake in the light increases linearly with increasing p O 2 while dark respiration is saturated at a p O 2 near 0.05 atm. Photorespiration is inhibited rapidly and completely by DCMU (3 x 10 -5 m) but KCN (10 -4 m) has little effect. Exogenously supplied hydroxyethane sulphonate (10 -5 m), an inhibitor of glycollate oxidase activity, and glycollate do not affect respiration, although 14 C-labelled glycollate is assimilated in the light and in the dark. Photorespiration is highly sensitive to p CO 2 and to NaHCO 3 concentration and approaches true photosynthetic oxygen production at the CO 2 compensation point of 10 parts/10 6 . A CO 2 concentration of 0.02 atm completely inhibits photorespiration whereas true photosynthesis is scarcely affected. Conditions which stimulate photorespiration (low p CO 2 and high p O 2 ) progressively inhibit acetylene reduction. In short-term studies DCMU inhibits acetylene reduction under condi­tions which stimulate photorespiration but has little effect under conditions which inhibit photorespiration. The results suggest that photorespiration and nitrogenase activity com­pete indirectly for reducing power and that at least one mechanism of oxygen inhibition of nitrogenase activity is via a stimulation of photorespiration.

scholarly journals Nutrient absorption by pea plants during dinitrogen fixation. 2. Effects of ambient acidity and temperature.

1982 ◽  
Vol 30 (2) ◽  
pp. 85-97
Author(s):  
M.L. van Beusichem
Keyword(s):  
Nutrient Uptake ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Dinitrogen Fixation ◽  
Proton Release ◽  
Proton Extrusion ◽  
Activity Unit ◽  
Anion Uptake ◽  
Automatic Titration

Nutrient uptake and biological dinitrogen fixation were studied using effectively nodulated pea plants which had been grown hydroponically for 6 wk in nutrient sol. of pH 4.0, 5.5 or 7.0. The pH of the root medium was kept constant by automatic titration and the temp. maintained at 13 deg C throughout. Although the N content of the plants grown at pH 4.0 and 7.0 was lower in comparison with that of plants of the pH 5.5 treatment, no N deficiency symptoms were observed. About the same number of nodules was formed in each of the treatments but they were smallest in the pH 5.5 treatment. Nevertheless, the total amount of dinitrogen fixed/plant at pH 5.5 was larger than that at higher or lower acidity. This was due to a higher nitrogenase activity/unit of nodule wt., which could be ascertained by an in vivo acetylene reduction test. In all cases more nutritive cations than anions were absorbed resulting in net proton extrusion by the roots. Both cation and anion uptake and hence the extent of proton release were affected substantially by ambient acidity. Plants grown at pH 7.0 generated 2.3 times as much acidity as plants grown at pH 4.0. The symbiotic dinitrogen fixing process was repressed by raising the temp. to 25 deg which resulted in no DM production and proton release and accelerated the onset of maturation. (Abstract retrieved from CAB Abstracts by CABI’s permission)

The effect of light-dependent oxygen consumption on nitrogenase activity in Anabaena cylindrica

1986 ◽  
Vol 64 (9) ◽  
pp. 1843-1848 ◽  
Author(s):  
Donald L. Smith ◽  
David G. Patriquin ◽  
Margareta Dijak ◽  
George M. Curry
Keyword(s):  
Oxygen Consumption ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Dark Respiration ◽  
Anabaena Cylindrica ◽  
Heat Treated ◽  
Carbonyl Cyanide ◽  
Photosynthetic Oxygen ◽  
Similar Decrease ◽  
Effect Of Light

Light-dependent oxygen consumption (LDOC) was observed in isolated heterocysts and in intact and sonicated CO2-fixing Anabaena cylindrica cells. The rate of LDOC in heterocysts was about three times that of CO2-fixing cells. Photosynthetic oxygen production by A. cylindrica became light saturated at 0.3 to 0.5 mW cm−2. LDOC and nitrogenase activity (acetylene reduction) increased with light intensity up to 2.5 mW cm−2 and incubation under air resulted in much larger relative acetylene reduction increases than incubation under N2. Carbonyl cyanide-m-chlorophenyl-hydrazone, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and cyanide did not affect the rate of LDOC in isolated heterocysts or cell-free preparations of CO2-fixing cells. However, all three substances induced LDOC in CO2-fixing cells. Heat treatment (100 °C for 1 min) caused a doubling of LDOC. Depletion of reduced carbon reserves by dark incubation caused a similar decrease in LDOC and dark respiration. The higher rates of LDOC observed in heat-treated materials were removed by catalase, but not by superoxide dismutase. Catalase injection released half of the O2 consumed through LDOC by heated preparations. LDOC increased with temperature up to 85 °C, and increased threefold with pH between pH 10 and 11.5. The possibility that LDOC may act to protect the nitrogenase of the heterocyst from oxygen inactivation is discussed.

Properties of the alfalfa–Rhizobium meliloti symbiotic nitrogenase enzyme system in vivo and in vitro

1986 ◽  
Vol 64 (6) ◽  
pp. 556-564 ◽  
Author(s):  
R. W. Miller ◽  
A. Al-Jobore ◽  
W. B. Berndt
Keyword(s):  
Reaction Temperature ◽  
Acetylene Reduction ◽  
Electron Flux ◽  
Rhizobium Meliloti ◽  
Enzyme System ◽  
Nitrogenase Activity ◽  
Adenylate Energy Charge ◽  
Temperature Range

A comparison of hydrogen production and acetylene reduction by excised alfalfa nodules, nitrogenase proteins isolated from Rhizobium meliloti bacteroids, and nitrogenase proteins purified from Klebsiella pneumoniae revealed several differences in the catalytic properties of the in vivo and in vitro systems. In all cases, assays for nitrogenase activity were carried out at near-physiological enzyme concentrations. Intact nodules maintained high nitrogenase activity to much lower reaction temperatures than the isolated enzymes. The Q10 value for acetylene reduction was 1.4 for nodules, but exceeded 5 for isolated R. meliloti nitrogenase in the temperature range 12–28 °C. The reduction of substrates requiring more than two reducing equivalents by the isolated enzyme system was severely restricted by the effects of reaction temperature on electron flux, as shown by very low values for electron allocation to dinitrogen determined indirectly at 12 °C. Temperature-induced lags in the initial reaction velocity of the reconstituted Klebsiella nitrogenase system were not observed with the isolated, but unseparated, R. meliloti nitrogenase proteins when acetylene was the substrate. The respiration-supported nodule system was not saturated with oxygen under air at reaction temperatures between 12 and 25 °C. Measurements of the adenine nucleotide levels of whole nodules indicated that the observed effects of reaction temperature on nitrogenase in vitro may be partially compensated in vivo by increases in adenylate energy charge and marked decreases in the ratio of ADP/ATP which occur as the nodule temperature is lowered. From the results reported here, it is clear that the R. meliloti – alfalfa nitrogenase system is limited in activity by oxygen supply. However, electron flux is sufficient within the physiological temperature range to maintain the Mo–Fe protein in the catalytically active state.

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

2018 ◽  
Author(s):  
Madushani Dharmarwardana ◽  
André F. Martins ◽  
Zhuo Chen ◽  
Philip M. Palacios ◽  
Chance M. Nowak ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Single Molecule ◽  
Biological Fluids ◽  
Cellular Respiration ◽  
Organic Radical ◽  
Paramagnetic Resonance ◽  
Metal Free ◽  
Disease States

Superoxide overproduction is known to occur in multiple disease states requiring critical care yet non-invasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by “click conjugating” paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced <i>in vivo</i> to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species—in particular superoxide—and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for <i>T<sub>1</sub></i> contrast at low field (<3.0 T), and <i>T<sub>2</sub></i> contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for “quenchless fluorescent” bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. <a>Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.</a>

scholarly journals Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
In Vitro Development ◽  
Porcine Islets ◽  
Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Estimating Nitrogenase Activity of Faba Bean (Vicia faba) by Acetylene Reduction (Ar) Assay

1990 ◽  
Vol 17 (5) ◽  
pp. 489 ◽  
Author(s):  
Herdina ◽  
JH Silsbury
Keyword(s):  
Diurnal Variation ◽  
Vicia Faba ◽  
Faba Bean ◽  
Closed System ◽  
N2 Fixation ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Electron Flow ◽  
Specific Rate ◽  
Rooting Medium

Methods of conducting acetylene reduction (AR) assay were appraised for estimating the nitrogenase activity of nodules of faba bean (Vicia faba L.). Factors considered were: (i) disturbance of plants when removing the rooting medium; (ii) assay temperature; (iii) the use of whole plants rather than detached, nodulated roots; (iv) diurnal variation in nodule activity; and (v) a decline in C2H4 production after exposure to C2H2. Plants growing in jars of 'oil dry' (calcined clay) had the same AR activity when assayed in situ in a closed system as when assayed after removal of the rooting medium. Assay temperatures of 12.5, 17.5 and 22.5°C influenced the specific rate of AR with the optimum at 17.5°C. Removal of the shoot resulted in a rapid decrease in AR activity in both vegetative and reproductive plants but the effect was much larger in the latter. AR and respiration by nodulated roots were closely linked and both varied markedly over a diurnal 12 h/12 h cycle. Since no fluctuation was found after nodules were detached, diurnal variation in the respiration of nodulated roots is attributed to change in nodule activity. Half of the dark respiration of nodulated roots was associated with respiration of the nodules and thus largely with N2 fixation. Since the AR assay provides no information on how electron flow in vivo is partitioned between reduction of N2 and reduction of protons, diurnal variation in hydrogen evolution (HE) in air and Ar/O2 in an open system was used to estimate this partitioning. Diurnal variation in apparent N2 fixation estimated in this manner was examined at a 'low' PPFD (300 μmol m-2 s-1) and at 'high' (1300 μmol m-2 s-1) to explore whether variation could be attributed to change in carbohydrate supply. Although HE in air and in Ar/O2 were both closely linked with the respiration of the nodulated root, apparent N2 fixation showed only a slight diurnal variation at 'low' light and almost none at 'high'. Vegetative plants showed no C2H2-induced decline in activity with exposure to C2H2 but reproductive plants did. This difference appears to be an age effect rather than attributable to flowering per se, since a decline occurred even when plants were kept vegetative by disbudding. A closed system for AR assay appears satisfactory for vegetative faba bean but such an assay over a 40-min period during the reproductive stage would underestimate nitrogenase activity by about 20%.

Dinitrogen fixation and nodulation by Frankia in Alnus incana as affected by inorganic nitrogen in pot experiments with peat

1983 ◽  
Vol 61 (11) ◽  
pp. 2956-2963 ◽  
Author(s):  
U. Granhall ◽  
T. Ericsson ◽  
M. Clarholm
Keyword(s):  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Inorganic Nitrogen ◽  
Experimental Period ◽  
Alnus Incana ◽  
Nitrogen Application ◽  
Noticeable Effect ◽  
Essential Nutrients ◽  
Pot Experiments ◽  
Ph Level

The effects of single large or repeated, exponentially increasing applications of nutrients, with or without inorganic nitrogen and at two pH levels, on the growth, nodulation, acetylene reduction, and nutrient uptake in Alnus incana (L.) Moench were investigated in pot experiments with peat under controlled laboratory conditions. The repeated application of inorganic nitrogen did not suppress nitrogenase activity until the last 2 weeks, whereas an initial, large, nitrogen application effectively inhibited nodulation and activity throughout the 40-day experimental period. The mode of nitrogen application was thus found to be more important than the total amounts applied. Shoot length, leaf area, shoot–root relations, dry-matter production, and nitrogen contents of plants were determined at the end of the experiment, as well as the effect of Frankia inoculations. Nitrogenase activity was determined three times, at 0, 3, and 5 weeks. N2 fixation (balance/acetylene reduction) was found to be maximal, 55% of total nitrogen uptake, in minus-N pots with single applications of essential nutrients. The fastest growth was, however, noted in pots with single applications of all nutrients, including N. Among the latter, pots inoculated with Frankia showed the best growth, in spite of low nitrogenase activity. The only noticeable effect of a raised pH level was a reduced endophyte activity in minus-N pots with single applications of essential nutrients, due to increased N mineralization in the peat.

Abstract 121: Development of Enzyme Replacement Therapy in Mammalian Models of Barth Syndrome

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Ana Dinca ◽  
Wei-Ming Chien ◽  
Michael Chin
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Cell Line ◽  
Single Gene ◽  
Barth Syndrome ◽  
Protein Therapy ◽  
Knock Out ◽  
Enzyme Replacement ◽  
C Terminus

Barth Syndrome (BTHS) is caused by a single gene mutation in the mitochondrial transacylase, tafazzin (TAZ), which results in impaired lipid metabolism leading to dysfunction in highly energetic tissues such as the heart and skeletal muscle. TAZ remodels the signature mitochondrial phospholipid, cardiolipin (CL), which is responsible for providing support to the electron transport chain. BTHS patients suffer from growth deficiencies, cardiomyopathy, hypotonia and neutropenia. Currently, treatment for patients with BTHS is supportive, seeking to ameliorate rather than prevent heart problems, skeletal muscle problems and recurring infections. Protein therapy, on the other hand, might treat and even prevent cardiac, skeletal muscle as well as infection-related morbidities. We designed a recombinant TAZ protein containing a cell penetrating peptide in its C-terminus, which enables the recombinant TAZ to penetrate cells and then treated TAZ-deficient cells with it. We tested the permeability of the recombinant protein by direct delivery to H9C2 cardiomyoblasts and found that the protein is successfully taken up by the cells. We have generated a CRISPR-mediated TAZ knock out cardiomyoblast cell line and we found that TAZ knock out cells show a decrease in oxygen consumption as compared to the wild type cells; this is consistent with data from BTHS patient-derived cells. We are using this cell line to assess the enzymatic activity of the delivered protein by conducting mitochondrial respiration measurements. We have also acquired a mouse model of BTHS and are testing the recombinant TAZ in vivo. Preliminary data shows an augmentation in oxygen consumption following treatment with TAZ. These results indicate that the protein is able to reach the mitochondria, where it is enzymatically active and able to enhance respiration. As the protein is able to rescue respiration in cells in which tafazzin was absent, this suggests that our approach should not only be able to prevent onset of symptoms, but also rescue the phenotype in already affected tissues.

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