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PubMed1995

Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: symmetry of the carbonyl interactions and close equivalence of the QB vibrations in Rhodobacter sphaeroides and Rhodopseudomonas viridis probed by isotope labeling.

The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis has been investigated by light-induced FTIR difference spectroscopy of RCs reconstituted with several isotopically labeled ubiquinones. The labels used were 18O on both carbonyls and 13C either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The QB-/QB spectra of RCs reconstituted with the isotopically labeled and unlabeled quinones as well as the double differences calculated from these spectra exhibit distinct isotopic shifts for a number of bands attributed to vibrations of QB and QB-. The vibrational modes of the quinone in the QB site are compared to those of ubiquinone in vitro, leading to band assignments for the C = O and C = C vibrations of the neutral QB and for the C***O and C***C of the semiquinone. The C = O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm-1 for both species. This demonstrates symmetrical and weak hydrogen bonding of the two C = O groups to the protein at the QB site. In contrast, the C = C vibrations are not equivalent for selective labeling at C1 or at C4, although they both contribute to the approximately 1617-cm-1 band in the QB-/QB spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C = C mode of QB does not involve displacement of the C4 carbon atom, while the motion of C1 is not hindered. Further analysis of the the spectra suggests that the protein at the binding site imposes a specific constraint on the methoxy and/or the methyl group proximal to the C4 carbonyl. The FTIR observations provide compelling evidence for almost identical conformation and identical interactions of the ubiquinone in the QB binding site of Rb. sphaeroides and Rp. viridis in contrast to the X-ray structures, which yield different descriptions for the hydrogen-bonding pattern of QB binding. In the semiquinone state, the bonding interactions of the C***O groups are also symmetrical and the C***C are inequivalent at C1 and C4. However, the interactions are almost the same in the RCs of both species.

Binding Sites↗

PubMed1976

The use of stable sulfur isotope labelling to elucidate sulfur metabolism by Clostridium pasteurianum.

An unique isotope labelling experiment was conducted whereby mixtures of sulfate and sulfite of different isotopic compositions were metabolized by Clostridium pasteurianum. The results showed during reduction of 1 mM SO3= plus 1 mM SO4=, essentially all evolved H2S arose from the sulfite whereas in the case of cellular sulfur, 85% was derived from sulfite and the remainder from sulfate.

Clostridium↗

PubMed2006

Thiazolidinedione bioactivation: a comparison of the bioactivation potentials of troglitazone, rosiglitazone, and pioglitazone using stable isotope-labeled analogues and liquid chromatography tandem mass spectrometry.

Troglitazone, a thiazolidinedione (TZD) type insulin sensitizer for the treatment of diabetes, was withdrawn from the U.S. market after several fatal cases of hepatotoxicity. Although the mechanism(s) of these idiosyncratic adverse reactions are not completely understood, circumstantial evidence suggests at least a partial contribution of reactive metabolite formation. Despite isolated case reports of hepatotoxicity, the other TZD derivatives pioglitazone and rosiglitazone are comparatively safe. Herein, we report on the bioactivation potential of these drugs and their TZD ring isotope-labeled 2-(15)N-3,4,5-(13)C(3) analogues in rat and human liver microsomes supplemented with glutathione (GSH). Screening for GSH adducts as surrogate markers for reactive intermediate formation was performed by liquid chromatography tandem mass spectrometry. Chemical characterization of the GSH conjugates was conducted by acquisition of their respective product ion spectra and the comparison between unlabeled and stable isotope-labeled TZD derivatives. The data suggest that all drugs undergo bioactivation processes via a common metabolic activation on the TZD ring, yielding disulfide type GSH conjugates as evidenced by the loss of labeled positions in the TZD moiety. Additional bioactivation processes leading to GSH adducts not involving TZD ring scission were evident for troglitazone. In human liver microsomes at low substrate concentrations, only troglitazone yielded a predominant GSH adduct not involving TZD ring scission. This property may contribute, together with other factors such as the relatively high dose administered as well as its potential to induce hepatic cholestasis and oxidative stress, to the hepatotoxicity of this drug.

Animals↗

PubMed2006

Efficient syntheses of four stable-isotope labeled (1R)-menthyl (1S,2S)-(+)-2-phenylcyclopropanecarboxylates.

Many carbenoid cyclopropanation reactions promoted by chiral catalysts give product mixtures reflecting impressive diastereo- and enantioselectivities. Few provide a single chiral product efficiently. This limitation has been overcome in cyclopropanations of styrene and isotopically labeled styrenes with alpha-diazoacetates. Convenient syntheses on a 20 g scale of each of four chiral isotopically labeled (1R)-menthyl (1S,2S)-2-phenylcyclopropanecarboxylates (the 1-d-3-(13)C, 1,(3S)-d2, 1,2,(3S)-d3, and 1,3,3-d3 isotopomers) of better than 99% ee have been realized.

Carbon Isotopes↗

PubMed1992

Exogenous substrate oxidation during exercise: studies using isotopic labelling.

This presentation summarizes the experimental data on the oxidation of exogenous substrates using isotopic labelling: glucose, fructose, maltodextrins, glucose polymers, starch and FFA. The two main determining factors for the oxidation of exogenous substrates are in this order: power output and amount of ingested substrates. The largest oxidation of exogenous substrate is observed for glucose, maltodextrins, Polycose, and starch. For exercises of one hour duration or more, the average rate of oxidation of these compounds can reach up to 0.5 g/min, which agrees with the indirect estimation of Coyle et al. Fructose is oxidized at a lower rate, except when it is taken before the exercise period. FFA are only oxidized to a small extent.

Carbon Isotopes↗

PubMed2003

Stable isotope labeling for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay analysis of ribonucleic acids.

Matrix-assisted laser desorption/ionization mass spectrometry is a powerful analytical tool for the structural characterization of oligonucleotides and nucleic acids. Here we report the application of stable isotope labeling for the simplified characterization of ribonucleic acids (RNAs). An (18)O label is incorporated at the 3'-phosphate of oligoribonucleotides during the enzymatic processing of intact RNAs. As implemented, a buffer solution containing a 50 : 50 mixture of H(2)O and (18)O-labeled H(2)O is used during endonuclease digestion. Upon digestion, characteristic doublets representative of the isotopic distribution of oxygen are noted for those products that contain 3'-phosphate groups. This approach is used to distinguish readily endonuclease digestion products from incomplete digestion products and non-specific cleavage products. In addition, RNase digestion products containing the characteristic isotopic doublet can be selected for further characterization by post-source decay (PSD) analysis. PSD products carrying the 3'-phosphate group will appear as a doublet, thereby simplifying fragment ion assignment.

Escherichia coli↗

PubMed1982

Assessment of glucose turnover in normal man with the use of a non-radioactive isotopically labelled preparation, [6,6-2H]glucose, as tracer.

1. Glucose kinetics were assessed in seven normal adult male subjects by an intravenous bolus technique with the use of a non-radioactive isotopically labelled preparation, [6,6-2H]glucose, as tracer. Tracer enrichment in plasma was assessed by gas chromatography-mass spectrometry. For comparison five subjects also received a simultaneous intravenous bolus of [6-3H]glucose and kinetics were assessed by conventional means. 2. Administration of [6,6-2H]glucose did not alter circulating glucose or insulin concentrations. 3. Glucose turnover, assessed by the use of [6,6-2H]glucose, was 11.4 (+/- 0.9) micromol min-1 kg-1 and 11.6 (+/- 0.5) micromol min-1 kg-1 with rate of glucose was 2.3 (+/- 0.3) ml min-1 kg-1 with both isotopically labelled tracers. Estimates of mean residence time, glucose pool and glucose space were also similar by each technique. 4. [6,6-2H]Glucose is therefore an effective tracer and allows investigation of glucose kinetics without administration of a radioactive label.

Adult↗

PubMed2002

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

3T3 Cells↗

PubMed2003

Biosynthesis of isotopically labeled gramicidins and tyrocidins by Bacillus brevis.

The three-dimensional structure of bilayer-associated gramicidin A is available from a structural data base. This and related peptides are, therefore, ideal model compounds to use during the implementation and development of new NMR techniques for the structural investigations of membrane proteins. As these methods rely on the isotopic labelling of single, selected or all sites, we have, investigated and optimised biochemical protocols using different strains of the Gram-positive bacterium Bacillus brevis. With newly developed schemes for isotopic labelling large amounts of gramicidin and tyrocidin enriched with stable isotopes such as (15)N or (15)N/(13)C have been obtained at low cost. A variety of analytical and spectroscopic techniques, including HPLC, mass spectrometry and NMR spectroscopy are used to characterise the resulting products.

Amino Acid Sequence↗

PubMed2004

Fractionation of stable isotope-labeled organic pollutants as a potential tracer of atmospheric transport processes.

To test the potential for using stable isotope fractionation to examine the atmospheric transport of semivolatile organic compounds (SOCs), we conducted simplified distillation experiments in the laboratory and a tracer-release experiment using mixtures of stable isotope-labeled (D and 13C) and unlabeled SOCs. Perdeuterated phenanthrene and alpha-hexachlorocyclohexane were transported more slowly via air-water gas exchange in our laboratory experiments, resulting in significant isotope fractionation of perdeuterated/unlabeled compound mixtures. In contrast, isotope fractionation of 13C6-labeled SOCs was much lower. A field tracer-release study was then conducted by spiking a seawater retention pond with a mixture of D10-labeled, 13C2-labeled, and unlabeled phenanthrene and examining isotope fractionation of the mixture after air-water gas exchange. No preferential fractionation of D10-vs 13C2-labeled phenanthrene was observed in the pond water; however, greater fractionation of D10-vs 13C2-labeled phenanthrene was observed in air samples collected within a 1-100 m radius of the pond. Thus, stable isotope tracers may provide a means of examining the atmospheric transport and air-earth exchange rates of POPs in an environmental realistic setting.

Air Movements↗

PubMed1975

A study of the mechanism of action of phosphorylase B using isotope-labelled substrates.

We have measured the alpha-deuterium and alpha-tritium secondary kinetic isotope effects on the synthesis and phosphorolysis of glycogen catalyzed by glycogen phosphorylase B (EC 2.4.1.1). The isotope-labeled substrates glucose-1T-1-phosphate and 1C14-glucose-1D-1-phosphate were used in the experiments, together with 6T-glucose-1-phosphate and C14-glucose-1-phosphate as controls. Measurement by the double label technique showed that both effects studied were equal to zero within the limits of experimental error. The possible mechanism of action of phosphorylase B is discussed.

Alkaline Phosphatase↗

PubMedDate not supplied

Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC).

We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.

Amino Acids↗

PubMed2002

Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry.

The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6(D) were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 (15)N-labeled and unlabeled ((14)N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [(15)N/(15)N] + [(15)N/(14)N] + [(14)N/(15)N] + [(14)N/(14)N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [(15)N] + [(14)N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).

Dimerization↗

PubMed2000

Isotopic labelling studies on far-infrared spectra of nickel-histamine complexes.

Nickel-histamine (hm) complexes type [Ni(hm)Cl2] and [Ni(hm)3]X2 (Where X= Cl, Br, I, ClO4) were investigated in the far-infrared region. Metal isotope labelling and deuteration effects were employed for observed band assignments. Metal-ligand vibrations were discussed and correlated with the structures of the complexes.

Histamine↗

PubMed2006

Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA.

Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems.

Amidohydrolases↗

PubMed2005

Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications.

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a tag into proteins for relative quantitation by mass spectrometry. SILAC is a simple, yet powerful, method for investigating the dynamics of protein abundance and posttranslational modifications. Here, we provide detailed instructions for using this method to study protein complexes, protein-protein interactions, and the dynamics of protein abundance and posttranslational modifications. We expect that SILAC will become a routine technique because of its applicability to most areas of cell biology. We have also developed a Web site (http://www.silac.org) to provide researchers with updated information about this method and related resources.

Amino Acids↗

PubMed2004

Stable isotope-labelled vitamin C as a probe for vitamin C absorption by human subjects.

Factors affecting absorption of physiological doses of vitamin C in man have not been widely studied, partly because few suitable tools exist to distinguish recently absorbed vitamin C from endogenous vitamin. Stable isotope-labelled vitamin C provides such a tool. Fifteen healthy non-smoking subjects aged 26-59 years were studied. Each received 30 mg l-[1-(13)C]ascorbic acid orally on two occasions, 3-4 weeks apart. The ascorbate was given alone or with Fe (100 mg as ferrous fumarate) or with red grape juice, which is rich in polyphenols. Blood was collected at frequent intervals for 1 h, and then each hour for a further 3 h. Total concentration of vitamin C was measured fluorometrically and its (13)C-isotope enrichment was measured by GC-MS after conversion to volatile trimethylsilyl esters. Peak plasma enrichment occurred within 25-50 min. No kinetic variables were significantly altered by the iron fumarate supplement. Grape juice attenuated vitamin C absorption, reaching significance at the 20 min time point. There were weak correlations between isotope enrichment and body weight or endogenous ascorbate concentration. The increment in total plasma ascorbate was smaller if calculated from isotope enrichment than from vitamin C concentration increase. The dilution pool was much larger than the plasma ascorbate pool. Further studies are needed to resolve these paradoxes. Stable isotope-labelled ascorbate is potentially useful for measurement of vitamin C absorption by human subjects.

Absorption↗

PubMed1987

Analytical methodology to determine stable isotopically labelled and unlabelled theophylline in human plasma using capillary gas chromatography-mass spectrometry.

A method is described for the simultaneous determination of [1,3-15N] theophylline and unlabelled theophylline in human plasma using gas chromatography-mass spectrometry. Plasma samples were subjected to extractive alkylation and the stable isotopically labelled and unlabelled forms of the drug were analysed as their N-pentafluorobenzyl derivatives on an SE-52 fused-silica capillary column. Quantitation was made by selected-ion monitoring employing as the internal standard 3-isobutyl-1-methylxanthine. The method has been used to study the absorption kinetics and bioavailability of a sustained release formulation of the drug when co-administered to human volunteers with a conventional formulation of the drug labelled with the stable isotope.

Adult↗