Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.     

New method of respiratory gas analysis: light spectrometer

A multigas concentration analyzer particularly suited for respiratory gas analysis has been developed using a new principle based on the measurement of the intensity of light emitted by excited atoms or ions in a direct current glow discharge. This glow discharge spectral emission gas analyzer (GDSEA), or light spectrometer, simultaneously measures O2, N2, CO2, He, and N2O gas concentrations with a 0-90% response time of 100 ms and a sample rate of less than 20 ml/min in a short gas sample line configuration. Mole accuracy and resolution of the GDSEA using a short sample line were determined in the laboratory to be +/- 0.15 to +/- 0.7% and 0.02-0.05%, respectively. In the clinical setting a comparative evaluation was made with a mass spectrometer in a long sample line, computerized, multibed, respiratory monitoring system. Results indicate a close agreement between the two instruments with differences in mixed inspiratory or expiratory O2 and CO2 concentrations of less than 2% and of derived variables, such as O2 consumption, CO2 production, and respiratory exchange ratio, of less than 5%.     

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.     

C4 photosynthetic isotope exchange in NAD-ME- and NADP-ME-type grasses

Monitoring photosynthetic isotope exchange is an important tool for predicting the influence of plant communities on the global carbon cycle in response to climate change. C(4) grasses play an important role in the global carbon cycle, but their contribution to the isotopic composition of atmospheric CO(2) is not well understood. Instantaneous measurements of (13)CO(2) (Delta(13)C) and C(18)OO (Delta(18)O) isotope exchange in five NAD-ME and seven NADP-ME C(4) grasses have been conducted to investigate the difference in photosynthetic CO(2) isotopic fractionation in these subgroups. As previously reported, the isotope composition of the leaf material (delta(13)C) was depleted in (13)C in the NAD-ME compared with the NADP-ME grasses. However, Delta(13)C was not different between subtypes at high light, and, although Delta(13)C increased at low light, it did so similarly in both subtypes. This suggests that differences in leaf delta(13)C between the C(4) subtypes are not caused by photosynthetic isotope fractionation and leaf delta(13)C is not a good indicator of bundle sheath leakiness. Additionally, low carbonic anhydrase (CA) in C(4) grasses may influences Delta(13)C and should be considered when estimating the contribution of C(4) grasses to the global isotopic signature of atmospheric CO(2). It was found that measured Delta(18)O values were lower than those predicted from leaf CA activities and Delta(18)O was similar in all species measured. The Delta(18)O in these C(4) grasses is similar to low Delta(18)O previously measured in C(4) dicots which contain 2.5 times the leaf CA activity, suggesting that leaf CA activity is not a predictor of Delta(18)O in C(4) plants.     

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is able to produce methane either from H2-CO2 or from formate. The route of formate entry into the methanogenic pathway was investigated by using 2H2O or [13C]formate and analysis by mass spectrometry. When cells (H2-CO2 or formate grown) were transferred to formate medium in 95% 2H water, the proportion of 2H in methane was 95%. When cells (H2-CO2 or formate grown) were transferred to media containing [13C]formate in the presence of H2-CO2 or He-CO2, the ratio of 13CH4 to 12CH4 increased over time parallel to the ratio of 13CO2 to 12CO2. The cells catalyzed a significant exchange of label between [13C]formate and 13CO2.     

A method to determine 18 O kinetic isotope effects in the hydrolysis of nucleotide triphosphates

A method to determine 18 O kinetic isotope effects (KIEs) in the hydrolysis of GTP that is generally applicable to reactions involving other nucleotide triphosphates is described. Internal competition, where the substrate of the reaction is a mixture of 18 O-labeled and unlabeled nucleotides, is employed, and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18 O at sites of mechanistic interest also contains 13C at all carbon positions, whereas the 16 O-labeled nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by the use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink interface. Carbon isotope ratios can be determined with accuracy and precision greater than 0.04% and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1(333)-catalyzed hydrolysis of [beta18 O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (<0.1%). A single KIE measurement can be conducted in 25 min with less than 5 microg nucleotide reaction product.     

Exchange of (18)O in the reaction of peroxynitrite with CO(2)

We have observed an exchange of (18)O in the reactions of CO(2) with peroxynitrite using membrane-inlet mass spectrometry and HPLC negative electrospray ionization mass spectrometry. The exchange appeared on addition of peroxynitrite to a solution containing (18)O-labeled CO(2) in equilibrium with bicarbonate. It was observed as a temporarily enhanced rate of depletion of (18)O from CO(2), a rate that was greater than the rate of (18)O depletion caused by the hydration/dehydration cycle of CO(2). In addition, we detected the appearance of mass peaks attributed to (18)O in product NO(3)(-).As a further measure of the (18)O exchange, there was a redistribution of (18)O such that the ratio of doubly to singly labeled CO(2) could not be described by the binomial expansion. This is not due to the hydration/dehydration cycle of CO(2) but most likely to recycling of CO(2) in the reaction with peroxynitrite. This (18)O exchange associated with the reactions of CO(2) and peroxynitrite may open a new methodology for studying this significant process.     

A rapid analytical technique for the determination of energy expenditure by the doubly labelled water method

The doubly labelled water method involves the administration of water enriched in 2H and 18O followed by determination of the turnover rates of these isotopes. Since 18O is eliminated from the body as both CO2 and water, while 2H leaves only as water, the difference between the two turnover rates provides a measure of CO2 production and hence energy expenditure. Isotopic analysis by conventional stable isotope ratio analysis (SIRA) is labour intensive and time consuming, as it requires off-line conversion of water samples to gases (H2 and CO2) followed by sequential analysis for each of the two isotopes using the mass spectrometer. Lack of suitable automated instrumentation with the ability to process large numbers of samples has prevented routine application of the method. We describe here an automated technique in which body water samples (urine, saliva, breath water or milk) are analysed simultaneously for 2H and 18O. The single bench system comprises two mass spectrometer analysers, one for measuring 2H from H2 gas, the other for measuring 18O from the water vapour (masses 18, 20). Both analysers share a common heated inlet system into which microlitre quantities of the body fluids are injected from an autosampler (102 samples). The water vapour flows both directly to one analyser for 18O measurement and into a uranium reduction furnace for conversion to H2, prior to 2H measurement by the second analyser. Both analysers also share vacuum and electronic components, enabling savings in both space and cost. In this paper we present results illustrating performance characteristics and procedures for routine application to human subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary diffusing capacities for nitric oxide and carbon monoxide determined by rebreathing in dogs

Pulmonary diffusing capacities (DL) of NO and CO were determined simultaneously from rebreathing equilibration kinetics in anesthetized paralyzed supine dogs (mean body wt 20 kg) after denitrogenation (replacement of N2 by Ar). During rebreathing the dogs were ventilated in closed circuit with a gas mixture containing 0.06% NO, 0.06% 13C18O, and 1% He in Ar for 15 s, with tidal volume of 0.5 liter and frequency of 60/min. The partial pressures of NO, 13C18O, 16O18O, N2, Ar, CO2, and He in the trachea were continuously analyzed by mass spectrometry. Measurements were performed at various O2 levels characterized by the mean end-expired PO2 during rebreathing (PE'O2). In control conditions ("normoxia," PE'O2 = 67 +/- 8 Torr) the following mean +/- SD values were obtained (in ml.min-1.Torr-1): DLNO = 52.4 +/- 11.0 and DLCO = 15.4 +/- 2.9. In hypoxia (PE'O2 = 24 +/- 7 Torr) DLNO increased by 11 +/- 8% and DLCO by 19 +/- 10%, and in hyperoxia (PE'O2 = 390 +/- 26 Torr) DLNO decreased to 87 +/- 3% and DLCO to 56 +/- 8% with respect to values in normoxia. DLNO/DLCO of 3.24 +/- 0.06 (hypoxia), 3.38 +/- 0.31 (normoxia), and 5.54 +/- 1.04 (hyperoxia) were significantly higher than the NO/CO Krogh diffusion constant ratio (1.92) predicted for simple diffusion through aqueous layers. With increasing O2 uptake elicited by 2,4-dinitrophenol, DLNO and DLCO increased and DLNO/DLCO remained close to unchanged. The results suggest that the combined effects of diffusion and chemical reaction with hemoglobin limit alveolar-capillary transport of CO. If it is assumed that reaction kinetics of NO with hemoglobin (known to be extremely fast) are not rate limiting for NO uptake, the contribution of the slow chemical reaction with hemoglobin to the total CO uptake resistance (= 1/DLCO) was estimated to be 38% in hypoxia, 41% in normoxia, and 64% in hyperoxia. The various factors expected to restrict the validity of this analysis are discussed, in particular the effects of functional inhomogeneity.     

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.     

Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 μl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r2 = 0.9998) over the concentration range of 1-1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34-110.67%. CPT13 in rat plasma was stable when stored at -20 °C or 4 °C for three freeze-thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.     

Noninvasive diffusing capacity and cardiac output in exercising dogs

We have developed a rebreathing procedure to determine diffusing capacity (DLCO) and pulmonary blood flow (Qc) in the awake, exercising dog. A low dead space, leak-free respiratory mask with an incorporated mouthpiece was utilized to achieve mixing between the rebreathing bag and the dog's lung. The rebreathing bag was initially filled with approximately 1.0 liter of gas containing 0.6% C2H2, 0.3% C18O, 9% He, and 35-40% O2. End-tidal gas concentrations were measured with a respiratory mass spectrometer. The disappearance of C2H2 and C18O was measured with respect to He to calculate Qc and DLCO. Values for DLCO in dogs, expressed per kilogram of body weight, were much larger than those reported in humans. However, at a given level of absolute O2 consumption, measurements of absolute DLCO in dogs were comparable to those reported in humans by both rebreathing and steady-state methods at rest and near-maximal exercise. These results suggest that DLCO is more closely matched to the metabolic capacity (i.e., maximal O2 consumption) than to body size between these two species.     

Membrane inlet ion trap mass spectrometry for the direct measurement of dissolved gases in ecological samples

The use of an ion trap mass spectrometer with three different membrane inlet probes is described. Two methods of removing water from the sample are compared. One is the use of a PTFE-silicone rubber double membrane, PTFE is relatively impermeable to water and so reduces the amount entering with the gas sample (Probe A). The second is the use of a silicone rubber membrane covered long probe, which condenses water out of the sample (Probe B). Response times (100%) for dissolved N2O, O2, Ar and CO2 without He in the chamber vary from between 158 and 684 s with Probe A. For the same probe with He, the response times were between 283 and 551 s. In the gas phase response times were between 99 and 153 s with He and 117 and 122 s without He. Probe B had 100% response of between 122 and 152 s for dissolved gases. Further extension of the probe by 2 m slowed response times as did increasing the ionisation time. Response times for Probe B increased to between 99 and 340 s when ionisation time increased from 1000 to 24,930 microseconds. Plots of output against concentration showed the steepest line of response for the short single membrane covered probe with 1000 microseconds ionisation time. Increasing the ionisation time, extending the probe and the use of a double membrane all reduced the gradient of output against concentration for every gas tested. In an intact sediment core, concentrations of O2, N2O and CO2 rose at the start and the concentration of N2 fell. As the disturbed sediment settled, this was reversed. The initial increase in O2 concentration stimulated respiration and inhibited the final pathway in dentrification producing higher concentrations of N2O and reducing the concentration of N2.     

Biodegradation of a biocide (Cu-N-cyclohexyldiazenium dioxide) component of a wood preservative by a defined soil bacterial community

The wood protection industry has refined their products from chrome-, copper-, and arsenate-based wood preservatives toward solely copper-based preservatives in combination with organic biocides. One of these is Cu-HDO, containing the chelation product of copper and N-cyclohexyldiazenium dioxide (HDO). In this study, the fate of isotope-labeled ((13)C) and nonlabeled ((12)C) Cu-HDO incorporated in wood sawdust mixed with soil was investigated. HDO concentration was monitored by high-pressure liquid chromatography. The total carbon and the δ(13)C content of respired CO(2), as well as of the soil-wood-sawdust mixture, were determined with an elemental analyzer-isotopic ratio mass spectrometer. The concentration of HDO decreased significantly after 105 days of incubation, and after 24 days the (13)CO(2) concentration respired from soil increased steadily to a maximum after 64 days of incubation. Phospholipid fatty acid-stable isotope probing (PFA-SIP) analysis revealed that the dominant PFAs C(19:0)d8,9, C(18:0), C(18:1)ω7, C(18:2)ω6,9, C(17:1)d7,8, C(16:0), and C(16:1)ω7 were highly enriched in their δ(13)C content. Moreover, RNA-SIP identified members of the phylum Acidobacteria and the genera Phenylobacterium and Comamonas that were assimilating carbon from HDO exclusively. Cu-HDO as part of a wood preservative effectively decreased fungal wood decay and overall microbial respiration from soil. In turn, a defined bacterial community was stimulated that was able to metabolize HDO completely.     

A new measurement technique reveals temporal variation in delta18O of leaf-respired CO2

The oxygen isotope composition of CO(2) respired by Ricinus communis leaves (delta(18)O(R)) was measured under non-steady-state conditions with a temporal resolution of 3 min using a tunable diode laser (TDL) absorption spectrometer coupled to a portable gas exchange system. The SD of delta(18)O measurement by the TDL was +/- 0.2 per thousand and close to that of traditional mass spectrometers. Further, delta(18)O(R) values at isotopic steady state were comparable to those obtained using traditional flask sampling and mass spectrometric techniques for R. communis grown and measured in similar environmental conditions. As well as higher temporal resolution, the online TDL method described here has a number of advantages over mass spectrometric techniques. At isotopic steady state among plants grown at high light, the "one-way flux" model was required to accurately predict delta(18)O(R). A comparison of measurements and the model suggests that plants grown under low-light conditions have either a lower proportion of chloroplast CO(2) that isotopically equilibrates with chloroplast water, or more enriched delta(18)O of CO(2) in the chloroplast that has not equilibrated with local water. The high temporal resolution of isotopic measurements allowed the first measurements of delta(18)O(R) when stomatal conductance was rapidly changing. Under non-steady-state conditions, delta(18)O(R) varied between 50 and 220 per thousand for leaves of plants grown under different light and water environments, and varied by as much as 100 per thousand within 10 min for a single leaf. Stomatal conductance ranged from 0.001 to 1.586 mol m(-2) s(-1), and had an important influence on delta(18)O(R) under non-steady-state conditions not only via effects on leaf water H(2) (18)O enrichment, but also via effects on the rate of the one-way fluxes of CO(2) into and out of the leaf.     

Photosynthetic Gas Exchange and Discrimination against 13CO2 and C18O16O in Tobacco Plants Modified by an Antisense Construct to Have Low Chloroplastic Carbonic Anhydrase

The physiological role of chloroplastic carbonic anhydrase (CA) was examined by antisense suppression of chloroplastic CA (on average 8% of wild type) in Nicotiana tabacum. Photosynthetic gas-exchange characteristics of low-CA and wild-type plants were measured concurrently with short-term, on-line stable isotope discrimination at varying vapor pressure deficit (VPD) and light intensity. Low-CA and wild-type plants were indistinguishable in the responses of assimilation, transpiration, stomatal conductance, and intercellular CO2 concentration to changing VPD or light intensity. At saturating light intensity, low-CA plants had lower discrimination against 13CO2 than wild-type plants by 1.2 to 1.8[per mille (thousand) sign]. Consequently, tissue of the low-CA plants was higher in 13C than the control plants. It was calculated that low-CA plants had chloroplast CO2 concentrations 13 to 22 [mu]mol mol-1 lower than wild-type plants. Discrimination against C18O16O in low-CA plants was 20% of that of the wild type, confirming a role of chloroplastic CA in the mechanism of discrimination against C18O16O ([delta]C18O16O). As VPD increased, stomatal closure caused a reduction in chloroplastic C02 concentration, and since VPD and chloroplastic CO2 concentration act in opposing directions on [delta]C18O16O, no effect of VPD was seen on [delta]C18O16O.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

根据光合作用能量代谢测定光呼吸的方法研究

本文根据光合作用和光呼吸途径能量代谢,通过改变外界CO2和O2浓度,计算卡尔文循环固定的CO2和光呼吸消耗的O2。结果表明,可以通过3种方法计算。方法1,测定在CO2饱和点（A）和正常CO2（A＇）浓度下吸收的CO2,得出光呼吸消耗的O2为：18／19（A-A＇）,卡尔文循环固定的CO2为：1／19（6A＋13A＇＋19Rd）。方法2,测定在不含O2的空气中（O）和正常O2（O’）浓度下释放的O2,得出光呼吸消耗的O2为：-13／5O-O＇-18／5Rd,卡尔文循环固定的CO2为：13／18（O＇—O）。方法3,测定在正常情况下吸收的CO2（A）和释放的O2（O＇）,得出光呼吸消耗的O2为：18（O＇—A＇）,卡尔文循环固定的CO2为：6O＇-5A＇＋Rd。测定在CO2饱和点和正常CO2浓度下吸收的CO2计算出水稻光呼吸释放的CO2占光合作用固定的24％-40％。     

A transgenic approach to understanding the influence of carbonic anhydrase on C18OO discrimination during C4 photosynthesis

The oxygen isotope composition of atmospheric CO(2) is an important signal that helps distinguish between ecosystem photosynthetic and respiratory processes. In C(4) plants the carbonic anhydrase (CA)-catalyzed interconversion of CO(2) and bicarbonate (HCO(3)(-)) is an essential first reaction for C(4) photosynthesis but also plays an important role in the CO(2)-H(2)O exchange of oxygen as it enhances the rate of isotopic equilibrium between CO(2) and water. The C(4) dicot Flaveria bidentis containing genetically reduced levels of leaf CA (CA(leaf)) has been used to test whether changing leaf CA activity influences online measurements of C(18)OO discrimination (Delta(18)O) and the proportion of CO(2) in isotopic equilibrium with leaf water at the site of oxygen exchange (theta). The Delta(18)O in wild-type F. bidentis, which contains high levels of CA relative to the rates of net CO(2) assimilation, was less than predicted by models of Delta(18)O. Additionally, Delta(18)O was sensitive to small decreases in CA(leaf). However, reduced CA activity in F. bidentis had little effect on net CO(2) assimilation, transpiration rates (E), and stomatal conductance (g(s)) until CA levels were less than 20% of wild type. The values of theta determined from measurements of Delta(18)O and the (18)O isotopic composition of leaf water at the site of evaporation (delta(e)) were low in the wild-type F. bidentis and decreased in transgenic plants with reduced levels of CA activity. Measured values of theta were always significantly lower than the values of theta predicted from in vitro CA activity and gas exchange. The data presented here indicates that CA content in a C(4) leaf may not represent the CA activity associated with the CO(2)-H(2)O oxygen exchange and therefore may not be a good predictor of theta during C(4) photosynthesis. Furthermore, uncertainties in the isotopic composition of water at the site of exchange may also limit the ability to accurately predict theta in C(4) plants.     

Molecular basis of the oxygen exchange from CO2 catalyzed by carbonic anhydrase III from bovine skeletal muscle

The exchange of 18O from CO2 to H2O in aqueous solution is caused by the hydration-dehydration cycle and is catalyzed by the carbonic anhydrases. In our previous studies of 18O exchange at chemical equilibrium catalyzed by isozymes I and II of carbonic anhydrase, we observed simple first-order depletion of 18O from CO2 with the 18O distribution among the species C18O18O, C16O18O, and C16O16O described by the binomial expansion (i.e., a random distribution of 18O). Using membrane-inlet mass spectrometry, we have measured 18O exchange between CO2 and H2O catalyzed by native zinc-containing and cobalt(II)-substituted carbonic anhydrase III from bovine skeletal muscle near pH 7.5. The distributions of 18O in CO2 deviate from the binomial expansion and are accompanied by biphasic 18O-exchange patterns; moreover, we observed regions in which 18O loss from CO2 was faster than 18O loss from HCO3-. These data are interpreted in terms of a model that includes 18O loss from an enzyme-substrate or intermediate complex. We conclude that more than one 18O can be lost from CO2 per encounter with the active site of isozyme III, a process that requires scrambling of oxygens in a bicarbonate-enzyme complex and cycling between intermediate complexes. This suggests that the rate of dissociation of H2(18)O (or 18OH-) from isozyme III is comparable to or faster than substrate and product dissociation.