Determination of substrate specificity of carboxylases by nuclear magnetic resonance

Determination of whether CO2 or HCO3- is the substrate for an enzymatic carboxylation has generally been accomplished by taking advantage of the fact that equilibration of these two compounds requires more than a minute at temperatures below 15 degrees C; thus different kinetics of carboxylation are obtained depending on whether CO2 or HCO3- is used to initiate the reaction. We report a new method using 13C18O2 as substrate for determining the CO2/HCO3- specificity of carboxylases. If CO2 is the substrate, then the 18O content of the 13C-containing product is the same as that of the 13CO2 used, whereas if HCO3- is the substrate, the 18O content is 2/3 that of the starting material. The method is independent of the detailed kinetics of the CO2/HCO3- interconversion and independent of the presence of contaminating unlabeled CO2 or HCO3-. Isotopic analysis is accomplished by 13C NMR. The method has been used to confirm that HCO3- is the substrate for phosphoenolpyruvate carboxylase. Studies of oxygen-18 isotope shifts in phosphorus NMR spectra have permitted confirmation of the observation that label is transferred from HC18O3- into Pi during the carboxylation of phosphoenolpyruvate.     

Radioisotopic assays of rates of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H2-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of 14CO by the bacterium was estimated at 38-56 micromol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm3 sediment per day. 85% of the utilized 14CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell bio mass production; and only just over 0.001% was converted to methane.     

The concentration and efflux of tree stem CO2 and the role of xylem sap flow

The accurate assessment of actual tree stem respiration and its relation with temperature plays a considerable role in investigating the forest carbon cycle.An increasing number of research reports have indicated that tree stem respiration determined with the commonlyapplied chamber gas exchange measuring system does not follow expectations regarding temperature relationships.theory that the respired CO2 in a tree stem would all diffuse outward into the atmosphere,However,it neglects partial CO2 that is dissolved in the xylem sap and is carried away by the transpirational stream.Scientists have started to realize that the respired CO2 measured with the chamber gas exchange method is only a portion of the total stem respiration (CO2 efflux),while the other portion,which is sometimes very substantial in quantity (thought to occupy maybe 15%-75% of the total stem respiration),is transported to the upper part of the stem and to the canopy by sap flow.This suggests that the CO2 produced by respiration is re-allocated within the stem.Accordingly,the change in CO2 efflux could be reflected in the rates of sap flow in addition to its dependence on temperature.Proper methods and instruments are required to quantify the internal and external CO2 fluxes in the trunk and their interaction with related environmental factors.     

Estimation of cardiac output by the CO2 rebreathing method during tethered swimming

The reproducibility of cardiac output (Q) estimated by the CO2 rebreathing method during tethered swimming was studied in five highly trained college swimmers. The reproducibility of the CO2 rebreathing method for estimations of Q during tethered swimming was similar to the reproducibility reported for the CO2 rebreathing method, direct Fick method, or dye-dilution method during either cycling or treadmill walking. All duplicate estimates of Q by the CO2 rebreathing method were within 15% of one another. A comparison was made between the Q's estimated by the CO2 rebreathing method during tethered swimming and previously published data on Q determined by the dye-dilution method during free swimming in a flune. At any given oxygen uptake, Q obtained by the CO2 rebreathing method during tethered swimming was not significantly different from the Q obtained by the dye-dilution method during flume swimming. Estimates of Q by the CO2 rebreathing method made during high intensities of tethered swimming were reproducible and appear to be valid.     

An analysis of estimation of pulmonary blood flow by the single-breath method

The single-breath method of determining pulmonary blood flow is a simple technique involving no inert gas or special maneuvers such as rebreathing or breath holding. The use of this elegant technique has been limited, however, largely because of questions regarding its accuracy. Previous analyses of the method have indicated that large errors in the estimated blood flow could result if data reduction is not handled carefully. In addition, an uncertain amount of error is introduced, if the CO2 retained by the lung tissue while measurements are being made is not taken into account in the calculations. This paper presents a rigorous approach for estimating the pulmonary blood flow by the single-breath method, which would minimize considerably the effects of measurement errors and would also allow for possible CO2 absorption by the lung tissue. It is based on the exact solution of the underlying equations that describe the dynamics of gas exchange in the lung. The analytic solution provides insight into the difficulties involved in extracting the desired information from the experimental data.     

A new approach to measure gross CO2 fluxes in leaves. Gross CO2 assimilation, photorespiration, and mitochondrial respiration in the light in tomato under drought stress

We developed a new method using 13CO2 and mass spectrometry to elucidate the role of photorespiration as an alternative electron dissipating pathway under drought stress. This was achieved by experimentally distinguishing between the CO2 fluxes into and out of the leaf. The method allows us to determine the rates of gross CO2 assimilation and gross CO2 evolution in addition to net CO2 uptake by attached leaves during steady-state photosynthesis. Furthermore, a comparison between measurements under photorespiratory and non-photorespiratory conditions may give information about the contribution of photorespiration and mitochondrial respiration to the rate of gross CO2 evolution at photosynthetic steady state. In tomato (Lycopersicon esculentum Mill. cv Moneymaker) leaves, drought stress decreases the rates of net and gross CO2 uptake as well as CO2 release from photorespiration and mitochondrial respiration in the light. However, the ratio of photorespiratory CO2 evolution to gross CO2 assimilation rises with water deficit. Also the contribution of re-assimilation of (photo) respiratory CO2 to gross CO2 assimilation increases under drought.     

A new two-breath technique for extracting the cerebrovascular response to arterial carbon dioxide

Cerebrovascular autoregulation is evaluated from spontaneous fluctuations in mean flow velocity (MFV) by transcranial Doppler ultrasound of the middle cerebral artery (MCA) with respect to changes in arterial blood pressure (BP(MCA)), but the effects of spontaneous fluctuations in arterial Pco(2) on MFV have been largely ignored. Autoregressive moving average analysis (ARMA), a closed-loop system identification technique, was applied to data from nine healthy subjects during spontaneous breathing, during inspiration of 10% CO(2) for two breaths once per minute for 4 min, and during sustained breathing of 7% CO(2). Cerebrovascular resistance index (CVRi) was calculated (CVRi = BP(MCA)/MFV). Reliable estimates of gain for BP(MCA) --> MFV were obtained for spontaneous breathing and the two-breath method. In contrast, reliable gain estimates for Pco(2) --> MFV or Pco(2) --> CVRi were achieved only under the two-breath method. Pco(2) --> MFV gain was smaller with the two-breath method than during sustained 7% CO(2) (P < 0.05). BP(MCA) was elevated by 7% CO(2) but not by the two-breath method. The closed-loop model provides insight into interactions between BP(MCA) and Pco(2) on cerebrovascular control, but reliable solutions for Pco(2) effects with ARMA analysis require perturbation by the two-breath method.     

A Mathematical Model for Leaf Photosynthesis of Mulberry

A mathematical model of leaf photosynthesis has been established. In this model, the processes of photosynthesis are divided into two parts, ie., the carboxylation process driven by light which is dependent on temperature and CO2 concentration, and the diffusion of CO2 from atmosphere to the carboxylation site. Finatly, CO2 uptake by the leaf is understood as dependent on 1), the CO2 response curve of the leaf mesophyll and 2). the CO2 partial pressure in the intercellular space in leaf. The COs response curve of the leaf photosynthesis is described mathematically in terms of carboxylation efficiency (Ca) or its initial slope and the photosynthetic capacity (Pm) or the CO2-saturated uptake rate of CO2 uptake, and dark respiration (Rd). The dependency of photosynthesis on leaf temperature and incident light intensity is incorporated into variations of those parameters which establish an appropriate response to internal CO2 pressure for particular light and temperature conditions prevailing at any time. Secondly the interactiion of stomata with photosynthesis is represented as an empirical relation between stomatal conductance and a combined environmental physiological index, APn·Hx/CaThe parameters used in the modelwere estimated with Marquardt-Newton method for non-linear function. Field measurements of mulberry leaf photosynthesis provided a data set for model testing. The resuks show that the simulated values of the model agree well with observed data. The model was used to analyse the response surface of leaf conductance and photosynthesis to environmental factors—Applications and limitations of the model are discussed     

Entrapment of carbon dioxide in the active site of carbonic anhydrase II

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO(2)-pressurized, cryo-cooled crystals to capture the first step of CO(2) hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO(2), for both the holo and the apo (without zinc) enzyme to 1.1A resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO(2) and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO(2) in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO(2). This method may also have a much broader implication for the study of other enzymes for which CO(2) is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.     

Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.     

Rapid Method for the Radioisotopic Analysis of Gaseous End Products of Anaerobic Metabolism

A gas chromatographic procedure for the simultaneous analysis of (14)C-labeled and unlabeled metabolic gases from microbial methanogenic systems is described. H(2), CH(4), and CO(2) were separated within 2.5 min on a Carbosieve B column and were detected by thermal conductivity. Detector effluents were channeled into a gas proportional counter for measurement of radioactivity. This method was more rapid, sensitive, and convenient than gas chromatography-liquid scintillation techniques. The gas chromatography-gas proportional counting procedure was used to characterize the microbial decomposition of organic matter in anaerobic lake sediments and to monitor (14)CH(4) formation from H(2) and (14)CO(2) by Methanosarcina barkeri.     

Optimization of the method of progressive hypercapnia by determination of the sensitivity threshold

Ventilatory response to CO2 rebreathing is a method which allows to evaluate the reactivity of chemoreceptors. However this method doesn't study the sensibility threshold, i.e. the Pe.t.CO2 value for which the ventilatory response appears clearly. This sensibility threshold was measured in 10 healthy subjects by rebreathing a gas mixture: 7% CO2 and 50% O2 to avoid hypoxy. It was defined as the value of Pe.t.CO2 for which the ventilation was above the tidal ventilation + 2 standard deviations. The sensibility threshold (51 +/- 4.35 mm Hg) was independent of the reactivity slope represented by the slope of the linear relation between minute ventilation (VE) and Pe.t.CO2 (1.34 +/- 0.60 l/min/mm Hg/m2) and consequently appears as an interesting parameter in order to evaluate the ventilatory response to CO2 by rebreathing.     

Utilization of a biogeochemical model in dendroecology. Application to the Cembro pine

Dendroecology, which is based on annual growth variable measurements, is in possession of data particularly well adapted to analyse the impact of global change on vegetation production. But the classical statistical methods of quantification of tree ring-climate relationship cannot take into account the effect of CO2 increase. Therefore, a biogeochemistry model (BIOME3) has been adapted to these data and then validated on Pinus cembra. The results indicate that the production is reduced by 14% if only the climatic changes are taken into account. If both climatic changes and CO2 increase are taken into account the production is increased by 62%. The direct fertilisation effect of CO2 increase will have more influence on the productivity than the indirect climatic effect.     

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H₂-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of ¹⁴CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm³ sediment per day. 85% of the utilized ¹⁴CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.     

A nondestructive method for the measurement of radioactive 14CO2 in blood

A method for estimation of 14CO2 present in blood and tissular samples is described. It is basically based on the introduction of large amounts of a gas mixture (95% O2, 5% CO2) in the samples which serves to remove the CO2 label by gas dilution. The gas phase is later captured in scintillation vials containing an organic-soluble base that retains the carbon label. The results obtained by means of this methodology show much better recoveries for blood samples than those obtained when the classic acid-diffusion method is used. In addition, it is a very fast procedure which does not alter the pH or protein integrity of the biological sample.     

The incorporation of carbon dioxide into the major classes of RNA during culture of the preimplantation mouse embryo.

The fixation of CO2 into major classes of RNA in the mouse embryo was studied in culture. Total fixation of CO2 was low at the two-cell stage and no label was found in RNA. Between the eight-cell and morula/early blastocyst stages of development, total fixation increased markedly but decreased again at the late blastocyst stage. On a per cell basis, the level of incorporation of CO2 decreased steadily throughout the preimplantation period. A significant acceleration in the accumulation of 14CO2 into all classes of RNA occurred between eight-celled embryos and morulae/early blastocysts, and this effect was more evident when results were calculated in relation to cell number. At the late blastocyst stage, incorporation of label into RNA decreased on a per embryo and a per cell basis. Most of the label from CO2 was incorporated into the r-RNA fraction at all stages of development and incorporation into s-RNA was always less. The pattern of labelling of RNA with 14CO2 was similar to that previously obtained for the incorporation of [3H]uridine into embryonic RNA, suggesting that most of the CO2 entering the RNA pool may be incorporated into nucleotide bases. The s-RNA and r-RNA fractions were susceptible to digestion with both pancreatic ribonuclease and 0-3 M alkali. Approximately 31% of the label in the TD-RNA fraction remained after hydrolysis with ribonuclease and a similar proportion of the TD-RNA was resistant to alkali treatment. Incorporation of CO2 by morulae/early blastocysts was substantial during culture in substrate-free medium but was increased significantly in medium containing lactate plus pyruvate. Carbon dioxide fixation into RNA was decreased by preculture for 48 hr before incubation in radioactive medium. When compared with freshly collected morulae/early blastocysts, the proportion of the total label in the s-RNA fraction of precultured embryos was low, and a correspondingly greater proportion of the total label was found in the TD-RNA fraction.     

Analysis of tricarboxylic acid-cycle metabolism of hepatoma cells by comparison of 14CO2 ratios.

The CO2-ratios method is applied to the analysis of abnormalities of TCA (tricarboxylic acid)-cycle metabolism in AS-30D rat ascites-hepatoma cells. This method utilizes steady-state 14CO2-production rates from pairs of tracers of the same compound to evaluate TCA-cycle flux patterns. Equations are presented that quantitatively convert CO2 ratios into estimates of probability of flux through TCA-cycle-related pathways. Results of this study indicated that the ratio of 14CO2 produced from [1,4-14C]succinate to 14CO2 produced from [2,3-14C]succinate was increased by the addition of glutamine (5 mM) to the medium. An increase in the succinate CO2 ratio is quantitatively related to an increased flux of unlabelled carbon into the TCA-cycle-intermediate pools. Analysis of 14C distribution in [14C]citrate derived from [2,3-14C]succinate indicated that flux from the TCA cycle to the acetyl-CoA-derived carbons of citrate was insignificant. Thus the increased succinate CO2 ratio observed in the presence of glutamine could only result from an increased flux of carbon into the span of the TCA cycle from citrate to oxaloacetate. This result is consistent with increased flux of glutamine to alpha-oxoglutarate in the incubation medium containing exogenous glutamine. Comparison of the pyruvate CO2 ratio, steady-state 14CO2 production from [2-14C]pyruvate versus [3-14C]pyruvate, with the succinate 14CO2 ratio detected flux of pyruvate to C4 TCA-cycle intermediates in the medium containing glutamine. This result was consistent with the observation that [14C]aspartate derived from [2-14C]pyruvate was labelled in C-2 and C-3. 14C analysis also produced evidence for flux of TCA-cycle carbon to alanine. This study demonstrates that the CO2-ratios method is applicable in the analysis of the metabolic properties of AS-30D cells. This methodology has verified that the atypical TCA-cycle metabolism previously described for AS-30D-cell mitochondria occurs in intact AS-30D rat hepatoma cells.     

Online measurement of dissolved carbon monoxide concentrations reveals critical operating conditions in gas fermentation experiments

Syngas fermentation is one possible contributor to the reduction of greenhouse gas emissions. The conversion of industrial waste gas streams containing CO or H₂, which are usually combusted, directly reduces the emission of CO₂ into the atmosphere. Additionally, other carbon‐containing waste streams can be gasified, making them accessible for microbial conversion into platform chemicals. However, there is still a lack of detailed process understanding, as online monitoring of dissolved gas concentrations is currently not possible. Several studies have demonstrated growth inhibition of Clostridium ljungdahlii at high CO concentrations in the headspace. However, growth is not inhibited by the CO concentration in the headspace, but by the dissolved carbon monoxide tension (DCOT). The DCOT depends on the CO concentration in the headspace, CO transfer rate, and biomass concentration. Hence, the measurement of the DCOT is a superior method to investigate the toxic effects of CO on microbial fermentation. Since CO is a component of syngas, a detailed understanding is crucial. In this study, a newly developed measurement setup is presented that allows sterile online measurement of the DCOT. In an abiotic experiment, the functionality of the measurement principle was demonstrated for various CO concentrations in the gas supply (0%–40%) and various agitation rates (300–1100 min^?1). In continuous stirred tank reactor fermentation experiments, the measurement showed reliable results. The production of ethanol and 2,3‐butanediol increased with increasing DCOT. Moreover, a critical DCOT was identified, leading to the inhibition of the culture. Thus, the reported online measurement method is beneficial for process understanding. In future processes, it can be used for closed‐loop fermentation control.     

Ventilatory response to inspired CO2 in the lizard, Tupinambis nigropunctatus

Elevating inspired levels of CO2 (1-4%) in Tupinambis nigropunctatus leads to an increase in tidal volume, mean expiratory flow, mean inspiratory flow, duration of the non-ventilatory period, inspiratory duration, expiratory duration and end inspiratory lung volume. Minute ventilation is variable and end expiratory volume decreases. An increase or decrease in CO2 concentration surrounding the head affects the duration of the non-ventilatory period before the altered CO2 concentration is inspired into the lungs. The change in duration of the non-ventilatory period before altered CO2 concentration is inspired into the lungs is probably mediated by CO2 sensitive receptors located in the mouth, nose or on the head surface.     

Effects of insulin on the pattern of glucose metabolism in the perfused working and Lagendorff heart of normal and insulin-deficient rats

1. The metabolic pattern of [U-(14)C]glucose in the isolated rat heart has been studied, with both retrograde aortic (Langendorff) and atrially (working) perfused preparations in the presence and absence of insulin, in normal animals, animals rendered insulin-deficient (by injection of anti-insulin serum 1hr. before excision of the heart) and animals rendered diabetic by streptozotocin injection 7 days before use. 2. Radioautochromatograms of heart extracts show that the pattern of glucose metabolism in heart muscle is more complex than in diaphragm muscle. In addition to (14)CO(2), glycogen, oligosaccharides, phosphorylated sugars and lactate (the main metabolites formed from [(14)C]glucose in diaphragm muscle), (14)C label from [(14)C]glucose appears in heart muscle in glutamate, glutamine, aspartate and alanine, and in tricarboxylic acid-cycle intermediates. 3. By a quantitative scanning technique of two-dimensional chromatograms it was found that a mechanical work load stimulates glucose metabolism, increasing by a factor of 2-3 incorporation of (14)C into all the metabolites mentioned above except lactate and phosphorylated sugars, into which (14)C incorporation is in fact diminished; (14)CO(2) production is equally stimulated. 4. Addition of insulin to the perfusion fluid of the working heart causes increases in (14)C incorporation, by a factor of about 1.5 into (14)CO(2), by a factor of about 3-5 into glycogen, lactate and phosphorylated sugars, by a factor of about 2-3 into glutamate and tricarboxylic acid-cycle intermediates and by a factor of about 0.5 into aspartate, whereas incorporation into alanine and glutamine is not affected. The effect of a work load on the pattern of glucose metabolism is thus different from that of insulin. 5. Increasing the concentration of glucose in the perfusion fluid from 1 to 20mm leads to changes of the pattern of glucose metabolism different from that brought about by insulin. (14)CO(2) production steadily increases whereas [(14)C]lactate and glycogen production levels off at 10mm-glucose, at values well below those reached in the presence of insulin. 6. In Langendorff hearts of animals rendered insulin-deficient by anti-insulin serum or streptozotocin, glucose uptake, formation of (14)CO(2) and [(14)C]lactate, and (14)C incorporation into glycogen and oligosaccharides are decreased. In insulin-deficient working hearts, however, glucose uptake and (14)CO(2) production are normal, whereas incorporation of (14)C into glycogen and [(14)C]lactate production are greatly decreased. 7. Insulin added to the perfusion fluid restores (14)C incorporation from glucose into (14)CO(2), glycogen and lactate in the Langendorff heart from animals rendered insulin-deficient by anti-insulin serum; in hearts from streptozotocin-diabetic animals addition of insulin restores (14)C incorporation into glycogen and lactate, but (14)CO(2) production remains about 50% below normal. 8. The bearing of these results on the problem of the mode of action of insulin is discussed.