Comparison of seasonal variations in water-use efficiency calculated from the carbon isotope composition of tree rings and flux data in a temperate forest

Tree-ring δ(13) C is often interpreted in terms of intrinsic water-use efficiency (WUE) using a carbon isotope discrimination model established at the leaf level. We examined whether intra-ring δ(13) C could be used to assess variations in intrinsic WUE (W(g), the ratio of carbon assimilation and stomatal conductance to water) and variations in ecosystem WUE (W(t) , the ratio of C assimilation and transpiration) at a seasonal scale. Intra-ring δ(13) C was measured in 30- to 60-μm-thick slices in eight oak trees (Quercus petraea). Canopy W(g) was simulated using a physiologically process-based model. High between-tree variability was observed in the seasonal variations of intra-ring δ(13) C. Six trees showed significant positive correlations between W(g) calculated from intra-ring δ(13) C and canopy W(g) averaged over several days during latewood formation. These results suggest that latewood is a seasonal recorder of W(g) trends, with a temporal lag corresponding to the mixing time of sugars in the phloem. These six trees also showed significant negative correlations between photosynthetic discrimination Δ calculated from intra-ring δ(13) C, and ecosystem W(t), during latewood formation. Despite the observed between-tree variability, these results indicate that intra-ring δ(13) C can be used to access seasonal variations in past W(t).     

Metabolic flux analysis in Synechocystis using isotope distribution from 13C-labeled glucose

Using the carbon isotope labeling technique, the response of cyanobacterial central carbon metabolism to the change in environmental conditions was investigated. Synechocystis was grown in the heterotrophic and mixotrophic cultures fed with 13C-labeled glucose. The labeling patterns of the amino acids in biomass hydrolysates for both cultures were detected by the two-dimensional 1H-13C correlation nuclear magnetic resonance (2D 1H-13C COSY NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) technique. The in vivo intracellular flux distributions were then quantitated from the labeling measurements and metabolite balances using a parameters fitting approach. From the estimated flux distributions, it was found that the pentose phosphate pathway was the major pathway of glucose catabolism in the heterotrophic culture, while in the mixotrophic culture, the flux of CO2 fixation through the Calvin cycle was about two-fold of the glucose input flux. The relative flux through the phosphoenolpyruvate carboxylase was very high in both cultures, and this reaction represented about 25% of the assimilated CO2 in the mixotrophic culture. More importantly, we found a substantial outflow from the tricarboxylic acid cycle to glycolysis pathway carried by the malic enzyme, demonstrating the operation of a C4 pathway in cyanobacterial cells through the PEP carboxylase and malic enzyme. The estimated flux distributions also revealed that the NADPH synthesis was in excess relative to its requirement, and the excess NADPH might be reoxidized in cyanobacterial respiration to provide the energy for cellular requirement. Moreover, the analyzed result also suggested that the activity of the respiratory electron transport chain in cyanobacterial cells was not inhibited by light.     

Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice.

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice.     

Calculation of isotope effects from first principles

Various means of calculating the effect of changing the mass of a given atom upon a chemical process are reviewed. Of particular interest is the deuterium isotope effect comparing the normal protium nucleus with its heavier deuterium congener. The replacement of the bridging protium in a neutral hydrogen bond such as the water dimer by a deuterium strengthens the interaction by a small amount via effects upon the vibrational energy. In an ionic H-bond such as the protonated water dimer, on the other hand, the reverse trend is observed in that replacement of the bridging protium by dimer weakens the interaction. In addition to the stability of a given complex, the rate at which a proton transfers from one group to another is likewise affected by deuterium substitution, viz. kinetic isotope effects (KIEs). The KIE is enlarged as the temperature drops, particularly so if the calculation of KIE includes proton tunneling. The KIE is also sensitive to any angular distortions or stretches present in the H-bond of interest. KIEs can be computed either by the standard transition state theory which is derived via only two points on the potential energy surface, or by more complete formalisms which take account of larger swaths of the surface. While more time intensive, the latter can also be applied to provide insights important in interpretation of experimental data.     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

13N isotope studies of glutamine assimilation pathways in Neurospora crassa.

L-[amide-13N]glutamine in Neurospora crassa is metabolized to [13N]glutamate by glutamate synthase and to [13N]ammonium by the glutamine transaminase-omega-amidase pathway. The [13N]ammonium released is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. Most of the nitrogen is retained during cycling between glutamate and glutamine.     

Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from ¹³C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of ¹³C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the ¹³C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the ¹³C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in ¹³C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in ¹³C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.     

Preservation effects on stable isotope ratios and consequences for the reconstruction of energetic pathways

Stable isotope analysis provides a powerful tool for describing the energetic pathways in a variety of ecosystems. However, isotope ratios of animal tissues can be altered by preservation methods, potentially leading to biased estimates of energy pathways when they are not taken into account. Here, we investigated the direct preservation effects of formalin, ethanol, NaCl, and drying on the δ¹³C and δ¹⁵N of fish muscle tissues, as well as the ultimate effects on the reconstruction of the energy pathways. All preservation methods, except drying, had significant impacts on δ¹³C and δ¹⁵N values. The effects of preservation appear to be highly taxa-specific and no significant time-dependent variations in nearly 2-year duration of preservation. δ¹³C and δ¹⁵N values were generally changed dramatically within the early stage of the preservation process and became stable over a relatively long-term preservation. Using an isotopic balance mixing model, the isotope-based food web reconstruction reveals that, without preservation correction, the importance of the pelagic energetic pathways for the fishes could be misestimated, except for the drying preservation. These results highlight that preservation can bias the interpretation of food web reconstruction results.     

Variation in fluxes estimated from nitrogen isotope discrimination corresponds with independent measures of nitrogen flux in Populus balsamifera L.

Acquisition of mineral nitrogen by roots from the surrounding environment is often not completely efficient, in which a variable amount of leakage (efflux) relative to gross uptake (influx) occurs. The efflux/influx ratio (E/I) is, therefore, inversely related to the efficiency of nutrient uptake at the root level. Time‐integrated estimates of E/I and other nitrogen‐use traits may be obtainable from variation in stable isotope ratios or through compartmental analysis of tracer efflux (CATE) using radioactive or stable isotopes. To compare these two methods, Populus balsamifera L. genotypes were selected, a priori, for high or low nitrogen isotope discrimination. Vegetative cuttings were grown hydroponically, and E/I was calculated using an isotope mass balance model (IMB) and compared to E/I calculated using ¹⁵N CATE. Both methods indicated that plants grown with ammonium had greater E/I than nitrate‐grown plants. Genotypes with high or low E/I using CATE also had similarly high or low estimates of E/I using IMB, respectively. Genotype‐specific means were linearly correlated (r = 0.77; P = 0.0065). Discrepancies in E/I between methods may reflect uncertainties in discrimination factors for the assimilatory enzymes, or temporal differences in uptake patterns. By utilizing genotypes with known variation in nitrogen isotope discrimination, a relationship between nitrogen isotope discrimination and bidirectional nitrogen fluxes at the root level was observed.     

Enzyme mechanisms from molecular modeling and isotope effects

The application of kinetic isotope effects and molecular modeling to characterize three enzyme-catalyzed reactions is presented; the mechanism of the chloroacid dehalogenase catalyzed reaction is approached using chlorine kinetic isotope effects and solvent kinetic isotope effects. The pre-steady-state phase of the reaction catalyzed by methylmalonyl-CoA mutase is approached by different QM/MM schemes and the results are validated by comparison with the experimental value of the deuterium kinetic isotope effect. Finally, a procedure for improving QM/MM calculations is illustrated by analysis of the trihydroxynaphthalene reductase-catalyzed reaction.     

Kinetics of metabolic pathways. A system in vitro to study the control of flux.

A method for determining Control Coefficients is proposed for systems studied in vitro and applied to a model pathway. Rat liver extract, which converts glucose into glycerol 3-phosphate, was used with the addition to the incubation mixture of fructose-bisphosphate aldolase, triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase as 'auxiliary' enzymes, which leaves all the control on the first three enzymes. The flux of the metabolic pathway was recorded by assaying NADH decay. Flux Control Coefficients (CJE) of hexokinase, glucose-6-phosphate isomerase and phosphofructokinase were calculated by titration of the system with increasing quantities of extraneous enzymes. It is shown that the summation property is fulfilled. The applicability of this procedure to study the control in any metabolic pathway is discussed. Possible relevance of the method to conditions in vivo and its limitations are considered.     

The effects of indomethacin on plasma LH levels in female and male deermice ( )

The purpose of this investigation was to determine (1) the approximate time of the preovulatory LH rise in cyclic deermice, (2) the effect of indomethacin administration on plasma LH levels during the expected preovulatory LH rise, and (3) the effect of indomethacin administration on plasma LH levels in castrated male deermice. The data indicate that the preovulatory LH rise occurred at about 2200 h on proestrus and that indomethacin pretreatment significantly reduced plasma LH levels during that time. In addition, indomethacin significantly reduced plasma LH levels in castrated male deermice. We conclude that plasma LH levels remain low throughout the estrous cycle with the exception of the preovulatory “surge”, that plasma LH levels in deermice are comparable to those reported for the rat, and that indomethacin pretreatment reduced plasma LH levels during periods when they were expected to be elevated (in castrated males and during the expected preovulatory LH rise in female deermice). The data are consistent with the hypothesis that the effect of indomethacin on plasma LH levels is due to an inhibitory effect on hypothalamic PG biosynthesis.     

Strategies for metabolic flux analysis in plants using isotope labelling

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.     

Deuterium isotope effects in mutagenesis by nitroso compounds.

Nitrosamines which have deuterium instead of hydrogen in the position alpha to the nitroso group have been reported to have reduced activity in carcinogenicity tests. This result implies that cleavage of a carbon--hydrogen bond is a limiting step in the reaction mechanism leading to tumor formation. Mutagenicity tests were undertaken with nitrosamines, which require metabolic activation, and with nitrosamides, which are directly acting mutagens, to determine the effect of deuterium substitution on the activity of each type of compound. Two nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine and methylnitrosourea) and three nitrosamines (dimethylnitrosamine, nitrosomorpholine, and dinitrosopiperazine) and their deuterium-containing analogs were tested for reversion of a nonsense mutation in the tyr locus of Escherichia coli WU 3610 (tyr-, leu-). Nitrosamines activated by rat-liver microsomes, but not nitrosamides, were less active as mutagens when the deuterium atom was present. The results suggest that the metabolic activation of nitrosamines to a mutagenic species involves the loss of hydrogen, a reaction which the nitrosamides, in the absence of enzyme, do not undergo.     

Kinetic isotope effects of nucleoside hydrolase from Escherichia coli

rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli (E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/Km kinetic isotope effects using uridine labeled with stable isotopes, such as 2H, 13C, and 15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012+/-0.006, 1.027+/-0.005, 1.134+/-0.007, 1.122+/-0.008, and 1.002+/-0.004 for [1'-13C], [1-15N], [1'-2H], [2'-2H], and [5'-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3'-exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata (C. fasciculata).     

Kinetic isotope effects in the photochemical cycle of bacteriorhodopsin.

Kinetics were determined for the four transients K590, L540, M410, O660 of the photochemical cycle of bacteriorhodopsin (BR570) both in 1H2O and in 2H2O over a wide temperature range. Breaks in the Arrhenius plots, observed at 25 degrees-32 degrees for the longest-lived transients coincide with a transition point in the microviscosity of the membrane as measured by depolarization of an added fluorescent probe. The earliest isotope effect occurs in the decay of L540, and is present in the subsequent formation and decay of M410 and O660. Thus in the light-driven proton pump of BR570, proton ejection from the Schiff base correlates with decay of L540 and reprotonation occurs with the decay of both M410 and O660 back to BR570.     

Inhibition of catalase-dependent ethanol metabolism in alcohol dehydrogenase-deficient deermice by fructose.

Some 40% of knee-joint synovial fluids from arthritic patients show the presence of bleomycin-detectable iron. This is released from a protein component of the fluid to bleomycin at acidic pH values. Patients whose fluids release iron have lower contents of transferrin, lactoferrin and caeruloplasmin than do patients whose fluids do not release iron to bleomycin. These proteins are important extracellular antioxidants, and measured antioxidant activities are extremely low in the iron-releasing fluids. The propensity of some fluids to release iron at low pH values, characteristic of the microenvironment beneath adherent macrophages, coupled with their decreased antioxidant protection against iron-stimulated oxygen-radical damage, might explain previously reported correlations between clinical disease severity, lipid peroxide content and the presence of bleomycin-detectable iron [Rowley, Gutteridge, Blake, Farr & Halliwell (1984) Clin. Sci. 66, 691-695].