Measurement of Photorespiratory Oxygen Exchange by Membrane Inlet Mass Spectrometry.

Photosynthesis and metabolism in plants involve oxygen as both a product and substrate. Oxygen is taken up during photorespiration and respiration and produced through water splitting during photosynthesis. To distinguish between processes that produce or consume O2 in leaves, isotope mass separation and detection by mass spectrometry allows measurement of evolution and uptake of O2 as well as CO2 uptake. This chapter describes how to calculate the rate of Rubisco oxygenation and carboxylation from in vivo gas exchange of stable isotopes of 16O2 and 18O2 with a closed cuvette system for leaf discs and membrane inlet mass spectrometry.

Measurement of O2 Uptake and Evolution in Leaves In Vivo Using Stable Isotopes and Membrane Inlet Mass Spectrometry.

Oxygen is both product and substrate of photosynthesis and metabolism in plants, by oxygen evolution through water splitting and uptake by photorespiration and respiration. It is important to investigate these processes simultaneously in leaves, especially in response to environmental variables, such as light and temperature. To distinguish between processes that evolve or take up O2 in leaves in the light, in vivo gas exchange of stable isotopes of oxygen and membrane inlet mass spectrometry is used. A closed-cuvette system for gas exchange of leaf discs is described, using the stable isotopes 16O2 and 18O2, with a semi-permeable membrane gas inlet and isotope mass separation and detection by mass spectrometry. Measurement of evolution and uptake, as well as CO2 uptake, at a range of light levels allows composition of a light response curve, here described for French bean (Phaseolus vulgaris) and maize (Zea mays) leaf discs.

Advances in Oxygen Isotope Analysis of Phosphate by Electrospray Orbitrap Mass Spectrometry for Studying the Microbial Metabolism of Microorganisms.

Understanding the impact of human activities on the metabolic state of soil and aquatic environments is of paramount importance to implement measures for maintaining ecosystem services. Variations of natural abundance 18O/16O ratios in phosphate have been proposed as proxies for the holistic assessment of metabolic activity given the crucial importance of phosphoryl transfer reactions in fundamental biological processes. However, instrumental and procedural limitations inherent to oxygen isotope analysis in phosphate and organophosphorus compounds have so far limited the stable isotope-based evaluation of metabolic processes. Here, we discuss how recent developments in Orbitrap high resolution mass spectrometry enable measurements of 18O/16O ratios in phosphate and outline the critical mass spectrometry parameters for accurate and precise analysis. Subsequently, we evaluate the types of 18O kinetic isotope effects of phosphoryl transfer reactions and illustrate how novel analytical approaches will give rise to an improved understanding of 18O/16O ratio variations from biochemical processes affecting the microbial phosphorus metabolism.

Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

Covariation between oxygen and hydrogen stable isotopes declines along the path from xylem water to wood cellulose across an aridity gradient.

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ18 O and δ2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ18 O and δ2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ2 H was well-correlated between leaf and branch, there was an offset in δ18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ18 O is used to reconstruct climatic scenarios. Conversely, comparing δ2 H and δ18 O patterns may reveal environmentally induced shifts in metabolism.

Unenriched xylem water contribution during cellulose synthesis influenced by atmospheric demand governs the intra-annual tree-ring δ18 O signature.

The oxygen isotope composition (δ18 O) of tree-ring cellulose is used to evaluate tree physiological responses to climate, but their interpretation is still limited due to the complexity of the isotope fractionation pathways. We assessed the relative contribution of seasonal needle and xylem water δ18 O variations to the intra-annual tree-ring cellulose δ18 O signature of larch trees at two sites with contrasting soil water availability in the Swiss Alps. We combined biweekly δ18 O measurements of soil water, needle water, and twig xylem water with intra-annual δ18 O measurements of tree-ring cellulose, xylogenesis analysis, and mechanistic and structural equation modeling. Intra-annual cellulose δ18 O values resembled source water δ18 O mean levels better than needle water δ18 O. Large parts of the rings were formed under high proportional exchange with unenriched xylem water (pex ). Maximum pex values were achieved in August and imprinted on sections at 50-75% of the ring. High pex values were associated with periods of high atmospheric evaporative demand (VPD). While VPD governed needle water δ18 O variability, we estimated a limited Péclet effect at both sites. Due to a variable pex , source water has a strong influence over large parts of the intra-annual tree-ring cellulose δ18 O variations, potentially masking signals coming from needle-level processes.

Hydrogen isotope fractionation in carbohydrates of leaves and xylem tissues follows distinct phylogenetic patterns: a common garden experiment with 73 tree and shrub species.

Recent methodological advancements in determining the nonexchangeable hydrogen isotopic composition (δ2 Hne ) of plant carbohydrates make it possible to disentangle the drivers of hydrogen isotope (2 H) fractionation processes in plants. Here, we investigated the influence of phylogeny on the δ2 Hne of twig xylem cellulose and xylem water, as well as leaf sugars and leaf water, across 73 Northern Hemisphere tree and shrub species growing in a common garden. 2 H fractionation in plant carbohydrates followed distinct phylogenetic patterns, with phylogeny reflected more in the δ2 Hne of leaf sugars than in that of twig xylem cellulose. Phylogeny had no detectable influence on the δ2 Hne of twig or leaf water, showing that biochemistry, not isotopic differences in plant water, caused the observed phylogenetic pattern in carbohydrates. Angiosperms were more 2 H-enriched than gymnosperms, but substantial δ2 Hne variations also occurred at the order, family, and species levels within both clades. Differences in the strength of the phylogenetic signals in δ2 Hne of leaf sugars and twig xylem cellulose suggest that the original phylogenetic signal of autotrophic processes was altered by subsequent species-specific metabolism. Our results will help improve 2 H fractionation models for plant carbohydrates and have important consequences for dendrochronological and ecophysiological studies.

16O/18O- exchange internal standard preparation enhancing reliability of bio-sample natural bioactive compounds absolute quantitation.

Natural bioactive compounds (NBCs) are regarded as candidates for many medical applications widely. Due to the complicated structure and biosynthesis source, only a few NBCs were supplied with commercial isotopic labeled standards. This shortage resulted in poor quantitation reliability in bio-samples for most NBCs, considering the remarkable matrix effects. NBCs metabolism and distribution studies would be restricted consequently. Those properties played critical roles in drug discovery and development. In this study, a fast, convenient, widely adopting 16O/18O exchange reaction was optimized for stable, available, affordable NBCs 18O-labeled standards preparation. With 18O- labeled internal standard, a UPLC-MRM-based NBCs pharmacokinetics analysis strategy was formed. Pharmacokinetics of caffeic acid with Hyssopus Cuspidatus Boriss extract (SXCF) dosed mice was carried out by established strategy. Compared with traditional external standards quantitation, adapting 18O-labeled internal standards, both accuracy and precision were enhanced significantly. Thus, the platform built by this work would accelerate the pharmaceutical research with NBCs, by providing a reliable, wide-adapted, affordable, isotopic internal standard-based bio-samples NBCs absolute quantitation strategy.

Isotope systematics of subfossil, historical, and modern Nautilus macromphalus from New Caledonia.

Cephalopod carbonate geochemistry underpins studies ranging from Phanerozoic, global-scale change to outcrop-scale paleoecological reconstructions. Interpreting these data hinges on assumed similarity to model organisms, such as Nautilus, and generalization from other molluscan biomineralization processes. Aquarium rearing and capture of wild Nautilus suggest shell carbonate precipitates quickly (35 µm/day) in oxygen isotope equilibrium with seawater. Other components of Nautilus shell chemistry are less well-studied but have potential to serve as proxies for paleobiology and paleoceanography. To calibrate the geochemical response of cephalopod δ15Norg, δ13Corg, δ13Ccarb, δ18Ocarb, and δ44/40Cacarb to modern anthropogenic environmental change, we analyzed modern, historical, and subfossil Nautilus macromphalus from New Caledonia. Samples span initial human habitation, colonialization, and industrial pCO2 increase. This sampling strategy is advantageous because it avoids the shock response that can affect geochemical change in aquarium experiments. Given the range of living depths and more complex ecology of Nautilus, however, some anthropogenic signals, such as ocean acidification, may not have propagated to their living depths. Our data suggest some environmental changes are more easily preserved than others given variability in cephalopod average living depth. Calculation of the percent respired carbon incorporated into the shell using δ13Corg, δ13Ccarb, and Suess-effect corrected δ13CDIC suggests an increase in the last 130 years that may have been caused by increasing carbon dioxide concentration or decreasing oxygen concentration at the depths these individuals inhabited. This pattern is consistent with increasing atmospheric CO2 and/or eutrophication offshore of New Caledonia. We find that δ44/40Ca remains stable across the last 130 years. The subfossil shell from a cenote may exhibit early δ44/40Ca diagenesis. Questions remain about the proportion of dietary vs ambient seawater calcium incorporation into the Nautilus shell. Values of δ15N do not indicate trophic level change in the last 130 years, and the subfossil shell may show diagenetic alteration of δ15N toward lower values. Future work using historical collections of Sepia and Spirula may provide additional calibration of fossil cephalopod geochemistry.

Metabolism is a major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra.

Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961-1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.

The effects on isotopic composition of leaf water and transpiration of adding a gas-exchange cuvette.

An expression was earlier derived for the non-steady state isotopic composition of a leaf when the composition of the water entering the leaf was not necessarily the same as that of the water being transpired (Farquhar and Cernusak 2005). This was relevant to natural conditions because the associated time constant is typically sufficiently long to ensure that the leaf water composition and fluxes of the isotopologues are rarely steady. With the advent of laser-based measurements of isotopologues, leaves have been enclosed in cuvettes and time courses of fluxes recorded. The enclosure modifies the time constant by effectively increasing the resistance to the one-way gross flux out of the stomata because transpiration increases the vapour concentration within the chamber. The resistance is increased from stomatal and boundary layer in series, to stomata, boundary layer and chamber resistance, where the latter is given by the ratio of leaf area to the flow rate out of the chamber. An apparent change in concept from one-way to net flux, introduced by Song, Simonin, Loucos and Barbour (2015) is resolved, and shown to be unnecessary, but the value of their data is reinforced.

A standard calculation methodology for human doubly labeled water studies.

The doubly labeled water (DLW) method measures total energy expenditure (TEE) in free-living subjects. Several equations are used to convert isotopic data into TEE. Using the International Atomic Energy Agency (IAEA) DLW database (5,756 measurements of adults and children), we show considerable variability is introduced by different equations. The estimated rCO2 is sensitive to the dilution space ratio (DSR) of the two isotopes. Based on performance in validation studies, we propose a new equation based on a new estimate of the mean DSR. The DSR is lower at low body masses (<10 kg). Using data for 1,021 babies and infants, we show that the DSR varies non-linearly with body mass between 0 and 10 kg. Using this relationship to predict DSR from weight provides an equation for rCO2 over this size range that agrees well with indirect calorimetry (average difference 0.64%; SD = 12.2%). We propose adoption of these equations in future studies.

Protein stable isotope probing with H2 18 O differentiated cold stress response at permissive temperatures from general growth at optimal conditions in Escherichia coli K12.

RATIONALE: Tracing isotopically labeled water into proteins allows for the detection of species-specific metabolic activity in complex communities. However, a stress response may alter the newly synthesized proteins. METHODS: We traced 18-oxygen from heavy water into proteins of Escherichia coli K12 grown from permissive to retardant temperatures. All samples were analyzed using UPLC/Orbitrap Q-Exactive-MS/MS operating in positive electrospray ionization mode. RESULTS: We found that warmer temperatures resulted in significantly (P-value < 0.05) higher incorporation of 18-oxygen as seen by both substrate utilization as relative isotope abundance (RIA) and growth as labeling ratio (LR). However, the absolute number of peptides with incorporation of 18-oxygen showed no significant correlation to temperature, potentially caused by the synthesis of different proteins at low temperatures, namely, proteins related to cold stress response. CONCLUSIONS: Our results unveil the species-specific cold stress response of E. coli K12 that could be misinterpreted as general growth; this is why the quantity as RIA and LR but also the quality as absolute number of peptides with incorporation (relative abundance, RA) and their function must be considered to fully understand the activity of microbial communities.

The oxygen isotopic signature of soil- and plant-derived sulphate is controlled by fertilizer type and water source.

The oxygen isotope signature of sulphate (δ18 Osulphate ) is increasingly used to study nutritional fluxes and sulphur transformation processes in a variety of natural environments. However, mechanisms controlling the δ18 Osulphate signature in soil-plant systems are largely unknown. The objective of this study was to determine key factors, which affect δ18 Osulphate values in soil and plants. The impact of an 18 O-water isotopic gradient and different types of fertilizers was investigated in a soil incubation study and a radish (Raphanus sativus L.) greenhouse growth experiment. Water provided 31-64% of oxygen atoms in soil sulphate formed via mineralization of organic residues (green and chicken manures) while 49% of oxygen atoms were derived from water during oxidation of elemental sulphur. In contrast, δ18 Osulphate values of synthetic fertilizer were not affected by soil water. Correlations between soil and plant δ18 Osulphate values were controlled by water δ18 O values and fertilizer treatments. Additionally, plant δ34 S data showed that the sulphate isotopic composition of plants is a function of S assimilation. This study documents the potential of using compound-specific isotope ratio analysis for investigating and tracing fertilization strategies in agricultural and environmental studies.

Seasonal and diurnal trends in progressive isotope enrichment along needles in two pine species.

The Craig-Gordon type (C-G) leaf water isotope enrichment models assume a homogeneous distribution of enriched water across the leaf surface, despite observations that Δ18 O can become increasingly enriched from leaf base to tip. Datasets of this 'progressive isotope enrichment' are limited, precluding a comprehensive understanding of (a) the magnitude and variability of progressive isotope enrichment, and (b) how progressive enrichment impacts the accuracy of C-G leaf water model predictions. Here, we present observations of progressive enrichment in two conifer species that capture seasonal and diurnal variability in environmental conditions. We further examine which leaf water isotope models best capture the influence of progressive enrichment on bulk needle water Δ18 O. Observed progressive enrichment was large and equal in magnitude across both species. The magnitude of this effect fluctuated seasonally in concert with vapour pressure deficit, but was static in the face of diurnal cycles in meteorological conditions. Despite large progressive enrichment, three variants of the C-G model reasonably successfully predicted bulk needle Δ18 O. Our results thus suggest that the presence of progressive enrichment does not impact the predictive success of C-G models, and instead yields new insight regarding the physiological and anatomical mechanisms that cause progressive isotope enrichment.

Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant.

Isotopic fractionation factors against 15N and 18O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ18O of water to investigate the effects of water δ18O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (15εNXR of -77.8 to -65.9‰ and 15ΔNXR of -31.3 to -30.4‰) and possibly O isotopic effect (18εNXR of -20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ18O vs δ15N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ18O vs δ15N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Spatial variations in the stable isotope composition of the benthic algae, Halimeda tuna, and implications for paleothermometry.

On Conch Reef, Florida Keys, USA we examined the effects of reef hydrography and topography on the patterns of stable isotope values (δ18O and δ13C) in the benthic green alga, Halimeda tuna. During the summer, benthic temperatures show high-frequency fluctuations (2 to 8 °C) associated with internal waves that advected cool, nutrient-rich water across the reef. The interaction between local water flow and reef morphology resulted in a highly heterogenous physical environment even within isobaths that likely influenced the growth regime of H. tuna. Variability in H. tuna isotopic values even among closely located individuals suggest biological responses to the observed environmental heterogeneity. Although isotopic composition of reef carbonate material can be used to reconstruct past temperatures (T(°C) = 14.2-3.6 (δ18OHalimeda - δ18Oseawater); r2 = 0.92), comparing the temperatures measured across the reef with that predicted by an isotopic thermometer suggests complex interactions between the environment and Halimeda carbonate formation at temporal and spatial scales not normally considered in mixed sediment samples. The divergence in estimated range between measured and predicted temperatures demonstrates the existence of species- and location-specific isotopic relationships with physical and environmental factors that should be considered in contemporary as well as ancient reef settings.

Metabolic incorporation of H218 O into specific glucose-6-phosphate oxygens by red-blood-cell lysates as observed by 13 C isotope-shifted NMR signals.

Water enriched with oxygen-18 (H218 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H218 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H218 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H218 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H218 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis.

Influence of Fermentation Water on Stable Isotopic D/H Ratios of Alcohol Obtained from Concentrated Grape Must.

According to Organisation Internationale de la vigne et du vin (OIV) standards, when analysing the stable isotope ratio of deuterium to hydrogen D/H at the methyl (I) and methylene (II) site of ethanol from concentrated must, a dilution with tap water is needed in order to carry out the alcoholic fermentation. This dilution causes a partial transfer of water hydrogens to the sugar, and this affects the (D/H)I and (D/H)II isotopic values of ethanol, which need to be normalised through specific equations based on the analysis of water δ18O or δ2H. The aim of this study was to evaluate the effectiveness and correctness of these equations experimentally. Grape, cane, and beet sugar, as well as grape must were diluted with water with increasing H and O stable isotope ratios, fermented, and analysed. SNIF-NMR and IRMS techniques were applied following the respective OIV methods. The equations based on the δ2H analysis of the diluted sugar/must solutions proved to be reliable in all the cases, although it is not an OIV standard. When using the equations based on the values of δ18O of the diluted solution, data normalisation was reliable only in cases where the water used for dilution had not undergone isotopic fractionation due, for example, to evaporation. In these cases, δ2H should be analysed.

Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation.

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18O atoms by deubiquitination in 18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18O-incorporation into ubiquitin.