Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus,as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - PEP phosphoenolpyruvate - RIE rocket immunoelectrophoresis - CIE crossed immunoelectrophoresis     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

The response of Synechococcus PCC7942 (Cyanophyta) to changes in CO2 supply in relation to the acclimation of the CO2-concentrating mechanism. I: physiological study

Distinct types of carboxysomes were distinguished in Synechococcus PCC 7942: electron-clear, electron-intermediate, carboxysomes with internal electron-clear areas, typical electron-dense and bar-shaped carboxysomes. Immunogold location with antibodies against the Rubisco large subunit showed specific label in all carboxysomes. The positive correlation between electron-density, the density of immunogold label, and the percentage of labeled structures within each type support a model of carboxysome biogenesis whereby electron-clear evolve to electron-intermediate and then to electron-dense carboxysomes by the progressive sequestering of Rubisco molecules. Cells responded to limitation in CO₂ supply by increasing carboxysome frequency and the proportion of typical electron-dense carboxysomes, the extent of the response depending on the degree of limitation. The time course of carboxysome expression during transfers between different conditions of CO₂ supply indicated that, under our experimental conditions, there were different levels of response, depending on the degree of limitation. The first level occured at atmospheric levels of CO₂ and involved changes in the affinity of the CCM and in carboxysome, which occurred simultaneously. More severe limitation of CO₂ supply affected carboxysomes exclusively, without further improvement in the affinity of the CCM.     

Clostridium methylpentosum sp. nov.: a ring-shaped intestinal bacterium that ferments only methylpentoses and pentoses

An intestinal bacterium isolated from a human subject utilized only two methylpentoses (L-rhamnose and L-fucose) and two pentoses (L-lyxose and D-arabinose) as fermentable substrates, among many compounds tested. The isolate was obligately anaerobic and had a distinctive morphology, its cells being rods bent in the shape of rings with the ends slightly overlapping. Single ring-shaped cells and left-handed helical chains of cells were present in cultures. The cells were surrounded by large capsules which appeared as thick, fibrous masses when examined by electron microscopy. Capsules were formed by cells growing in media containing any one of the four fermentable substrates. Terminally located, heat-resistant endospores were formed on plates of an enriched agar medium supplemented with L-rhamnose. End products of L-rhamnose or L-fucose fermentation included acetate, propionate, n-propanol, CO₂, and H₂. The isolate represented a new species of Clostridium for which the name Clostridium methylpentosum (type strain R2. ATCC 43829) is proposed. This organism may participate in intestinal digestive processes by metabolizing rhamnose released via the enzymatic depolymerization of dietary pectin.Abbreviations G+C guanine plus cytosine - OD optical density - TEM transmission electron micrograph     

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA₃-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA₃-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Regulation of photosynthetic electron-transport in Phaseolus vulgaris L., as determined by room-temperature chlorophyll a fluorescence

The regulation of photosystem II (PSII) by light-, CO₂-, and O₂-dependent changes in the capacity for carbon metabolism was studied. Estimates of the rate of electron transport through PSII were made from gas-exchange data and from measurements of chlorophyll fluorescence. At subsaturating photon-flux density (PFD), the rate of electron transport was independent of O₂ and CO₂. Feedback on electron transport was observed under two conditions. At saturating PFD and low partial pressure of CO₂, p(CO₂), the rate of electron transport increased with p(CO₂). However, at high p(CO₂), switching from normal to low p(O₂) did not affect the net rate of photosynthetic CO₂ assimilation but the rate of electron-transport decreased by an amount related to the change in the rate of photorespiration. We interpret these effects as 1) regulation of ribulose-1,5-bisphosphatecarboxylase (RuBPCase, EC 4.1.1.39) activity to match the rate of electron transport at limiting PFD, 2) regulation of electron-transport rate to match the rate of RuBPCase at low p(CO₂), and 3) regulation of the electron-transport rate to match the capacity for starch and sucrose synthesis at high p(CO₂) and PFD. These studies provide evidence that PSII is regulated so that the capacity for electron transport is matched to the capacity for other processes required by photosynthesis, such as ribulose-bisphosphate carboxylation and starch and sucrose synthesis. We show that at least two mechanisms contribute to the regulation of PSII activity and that the relative engagement of these mechanisms varies with time following a step change in the capacity for ribulose-bisphosphate carboxylation and starch and sucrose synthesis. Finally, we take advantage of the relatively slow activation of deactivated RuBPCase in vivo to show that the activation level of this enzyme can limit the rate of electron transport as evidenced by increased feedback on PSII following a step change in p(CO₂). As RuBPCase as activated, the feedback on PSII declined.Abbreviations and symbols J_C electron-transport rate calculated from CO₂-assimilation measurements - J_F electron-transport rate calculated from fluorescence parameters - PFD photon-flux density - q_E energy-dependent quenching - PSII photosystem II - q_Q Q-dependent quenching - QY quantum yield - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) C.I.W. publication No. 1015     

Allohydroxy-d-proline dehydrogenase

Growth of Pseudomonas aeruginosa PA01 on isomers of hydroxyproline induced the synthesis of an allohydroxy-d-proline dehydrogenase. The enzyme resembled the d-alanine dehydrogenase of this organism in its association with the particulate fraction and its linkage to oxygen through a cytochrome-containing respiratory chain, but differed from this and other bacterial d-amino acid dehydrogenases in its high substrate specificity and low K _m .     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Land plants of amphibious Littorella uniflora (L.) Aschers. maintain utilization of CO2 from the sediment

Summary Submerged macrophytes of the isoetid life form derive the majority of their CO₂ for photosynthesis from the sediment. The experiments described here were designed to test the hypothesis that root uptake of CO₂ is important also in the terrestrial form of Littorella uniflora. The results of ¹⁴CO₂ experiments showed that sediment CO₂ contributed 56% of the total fixation at 0.1mm CO₂ in the rhizosphere, 83% at 0.5mm and 96% at 2.5mm. Sediment CO₂ in emergent Littorella stands ranged from 0.1 to 1.0mm and averaged 0.5mm. Measurements of the net CO₂ exchange over the leaves showed an even higher dependence of the sediment as CO₂ source. Littorella leaves had no stomata at the base and densities (ca. 100 mm^–2) typical of terrestrial plants at the tip, allowing sediment-derived CO₂ to be supplied along the length of the leaf. The stomata permit supply of CO₂ from the air during periods of reduced sediment CO₂ concentrations (e.g. if the sediment dries up) and regulate transpiration.     

A novel mechanism involved in the metabolism of the tartaric acid stereoisomers in Rhodopseudomonas sphaeroides: enzymatic conversion of meso-tartaric acid to D(-)-glyceric acid and CO2

In cell extracts of Rhodopseudomonas sphaeroides grown on meso-tartrate the activities of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and 1.1.1.83, respectively) could be measured spectrophotometrically but not the activity of a meso-tartrate dehydrogenase or dehydratase. However, an enzyme activity was detected manometrically that catalyzed the stoichiometric release of CO₂ from mesotartrate in a molar ratio of 1:1. This reaction required catalytic amounts of NAD and the presence of both divalent (Mn²⁺ or Mg²⁺) and monovalent (NH ₄ ⁺ or K⁺) cations. Purification of the meso-tartrate decarboxylase showed that it was part of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating), which thus possessed a third catalytic function. The homogeneous enzyme catalyzed the stoichiometric conversion of incso-tartaric acid to D(-)-glyceric acid and CO₂. All interfering catalytic activities had been eliminated during the course of enzyme purification.     

Lymphocyte subsets and T(h)1/T(h)2 immune responses in patients with adenocarcinoma of the oesophagus or oesophagogastric junction: relation to pTNM stage and clinical outcome

Introduction Recent studies have indicated that the cytokines produced by CD4⁺ T helper type 1 (T_h1) and type 2 (T_h2) cells are critically important in antitumour immunity and perhaps clinical outcome. From this perspective, we investigated the immunocompetence of patients with previously untreated cancer of the oesophagus or oesophagogastric junction (OGJ) in relation to stage of disease and postoperative survival.Methods Blood samples were taken prior to surgery from 32 patients with adenocarcinoma of the oesophagus or OGJ. Ten healthy volunteers served as normal controls. T-cell and monocyte subpopulations were determined using flow cytometry. Monocyte as well as T_h1- and T_h2-lymphocyte cytokine levels were assessed in stimulated whole blood cultures.Results Absolute T-cell and monocyte (subset) counts as well as monocyte cytokine levels were similar among patients and controls. Production of T_h1-type cytokines was higher in patients than in controls (IFN-, p=0.01; IL-2, p=0.05), whereas T_h2-type cytokine levels were comparable (IL-4, p=0.5; IL-13, p=0.3). T-cell CD4⁺/CD8⁺ ratios decreased as pTNM stage worsened (stage I/II vs stage III/IV, p=0.009). Of all measured immunological parameters, only IL-2 production significantly affected both overall survival (p=0.015) and disease-free survival (p=0.0062). High IL-2 levels corresponded with a favourable prognosis.Conclusions Patients awaiting surgery for adenocarcinoma of the oesophagus or oesophagogastric junction demonstrated a shift in the T_h1/T_h2 balance—in favour of T_h1—compared with healthy volunteers. The ability of T cells to produce IL-2 was related to survival indicating a crucial role of T_h1-type cells in antitumour immunosurveillance.     

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of β-d-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid β-d-galactosidase with a low mobility and maximal activity atpH 3–5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-d-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 → 4)-lactone. (2) Lactose-hydrolyzing β-d-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active atpH 5–6 and strongly inhibited by glucono-(1 → 5)-lactone but not much affected by galactono-(1 → 4)-lactone. (3) Neutral β-d-galactosidase with a fast mobility and maximal activity atpH 6–8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-β-d-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 → 5)-lactone and (less) by galactone-(1 → 4)-lactone. Neutral β-d-galactosidase and neutral β-d-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral β-d-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation. This investigation was supported in part by Public Health Service Grant RR-00251 from the Division of Research Resources and by funds of the University of Utrecht.     

Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH ₄ ⁺ to the medium. The glutamate analog,l-methionine-dl-sulfoximine (MSX), derepressed N₂ fixation even in the presence of 10 mM extracellular NH ₄ ⁺ . When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N₂ as NH ₄ ⁺ . Nitrogenase activities and NH ₄ ⁺ production from fixed N₂ were increased considerably when a combined nitrogen source, NH ₄ ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH ₄ ⁺ as well as by glutamate.The results demonstrate that utilization of solar energy to photoproduce large quantities of NH ₄ ⁺ from N₂ is possible with photosynthetic bacteria by interfering with their regulatory control of N₂ fixation.     

Activation of cyclooxygenases by H2O2and t-butylhydroperoxide in aged rat lung

The arachidonate cascade is important for the generation of reactive species (RS), and cyclooxygenase (COX) is a key enzyme of this cascade. Tissues of 24-month-old rat lung showed a 2-fold increase in RS, malondialdehyde and thromboxane B₂ than those of 6-month-old rat. We found that the effects of 50 µM H₂O₂ and 200 µM t-butylhydroperoxide (t-BHP) specify on COX activity, and that their effects increased cytosolic COX activity in a concentration-dependent manner (1–50 µM) in 24-month-old rat. Our results suggested that COX activators such as t-BHP and H₂O₂, which are located in cytosol, are essential for the activation of COX in aged lung.     

Effect of methanogenic substrates on coenzyme F420-dependent N5,N10-methylene-H4MPT dehydrogenase,N5,N10-methenyl-H4MPT cyclohydrolase and F420-reducing hydrogenase activities in Methanosarcina barkeri

We measured F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydro-methanopterin dehydrogenase, N⁵, N¹⁰-methenyltetrahydro-methanopterin cyclohydrolase, and F₄₂₀-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H₂–CO₂-, methanol-, and H₂–CO₂+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H₂–CO₂, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H₂–CO₂- or methanol-grown cells, acetate-or H₂–CO₂ + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO₂-reduction pathway operated in the reverse direction. 2. When steps from CO₂ to CH₃-S-CoM in the CO₂-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered.Abbreviations MF methanofuran - H₄MPT 5,6,7,8-tetrahydromethanopterin - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP heterodisulfide of HS-CoM and HS-HTP - F₄₂₀ coenzyme F₄₂₀ (a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative) - H₂F₄₂₀ reduced coenzyme F₄₂₀ - HC⁺=H₄MPT N⁵,N¹⁰-methenyl-H₄MPT - H₂C=H₄MPT N⁵,N¹⁰-methylene-H₄MPT - H₃C=H₄MPT N⁵-methyl-H₄MPT - BES 2-bromoethanesulfonic acid     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.     

The relationship between carbon-dioxide-limited photosynthetic rate and ribulose-1,5-bisphosphate-carboxylase content in two nuclear-cytoplasm substitution lines of wheat,and the coordination of ribulose-bisphosphate-carboxylation and electron-transport capacities

Photosynthesis in two cultivars of Triticum aestivum was compared with photosynthesis in two lines having the same nuclear genomes but with cytoplasms derived from T. boeoticum. The in-vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) isolated from lines with T. boeoticum cytoplasm was only 71% of that of normal T. aestivum. By contrast, the RuBPCase activities calculated from the CO₂-assimilation rate at low partial pressures of CO₂, p(CO₂), were the same for all lines for a given RuBPCase content. This indicates that both types of RuBPCase have the same turnover numbers in-vivo of 27.5 mol CO₂·(mol enzyme)^–1·s^–1 (23°). The rate of CO₂ assimilation measured at normal p(CO₂), p _a =340 bar, and high irradiance could be quantitatively predicted from the amount of RuBPCase protein. The maximum rate of RuBP regeneration could also predict the rate of CO₂ assimilation at normal ambient conditions. Therefore, the maximum capacities for RuBP carboxylation and RuBP regeneration appear to be well-balanced for normal ambient conditions. As photosynthetic capacity declined with increasing leaf age, the capacities for RuBP carboxylation and RuBP regeneration declined in parallel.Abbreviations PAR photosynthetically active radiation - RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta)

In order to investigate the possible impacts of increased atmospheric CO₂ levels on algal growth and photosynthesis, the influence of CO₂ concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO₂ concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 M CO₂ in C. reinhardtii, C. pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO₂ was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N inC. pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p < 0.05) in high-CO₂-grown cells. Elevation of the CO₂ levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C. pyrenoidosa and S. obliquus when exposed to high photon flux density. The photoinhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO₂-grownC. pyrenoidosa and S. obliquus. Although pH and pCO₂ effects cannot be distinguished from this study, it can be concluded that increased CO₂ concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus.