[Protein metabolism of the female genital organs. Radioautographic research using isotope-labelled amino acids in female rats].
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Amino Acids↗
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Analysis of flash-induced FTIR difference spectra of the S-state cycle in the photosynthetic water-oxidizing complex by uniform 15N and 13C isotope labeling.
Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform (15)N and (13)C isotopic labeling of photosystem II (PS II). The difference spectra upon first- to fourth-flash illumination were obtained with hydrated (for the 1800-1200 cm(-)(1) region) or deuterated (for the 3500-3100 cm(-)(1) region) films of unlabeled, (15)N-labeled, and (13)C-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon (15)N and (13)C labeling provided the assignments of major peaks in the regions of 3450-3250 and 1700-1630 cm(-)(1) to the NH stretches and amide I modes of polypeptide backbones, respectively, and the assignments of some of the peaks in the 1600-1500 cm(-)(1) region to the amide II modes of backbones. Other prominent peaks in the latter region and most of the peaks in the 1450-1300 cm(-)(1) region exhibited large downshifts upon (13)C labeling but were unchanged by (15)N labeling, and hence assigned to the asymmetric and symmetric COO(-) stretching vibrations, respectively, of carboxylate groups in Glu, Asp, or the C-terminus. Peak positions corresponded well with each other among the first- to fourth-flash spectra, and most of the bands in the first- and/or second-flash spectra appeared with opposite signs of intensity in the third- and/or fourth-flash spectra. This observation indicates that the protein movements in the S(1)-->S(2) and/or S(2)-->S(3) transitions are mostly reversed in the S(3)-->S(0) and/or S(0)-->S(1) transitions, representing a catalytic role of the protein moieties of the water-oxidizing complex. Drastic structural changes in carboxylate groups over the S-state cycle suggest that the Asp and/or Glu side chains play important roles in the reaction mechanism of photosynthetic water oxidation.
Binding Sites↗
A mass spectrometric method for the determination of stable isotope labeled phenytoin suitable for pulse dosing studies.
A gas chromatographic mass spectrometric method has been developed for the determination in biological fluids of phenytoin (5,5-diphenylhydantoin), 5-(4-hydroxyphenyl)-5-phenylhydantoin (the major metabolite of phenytoin), and simultaneously, their stable isotope labeled analogs [5,5-diphenyl-2-13C-1,3-15N2-hydantoin and 5-(4-hydroxyphenyl)-5-phenyl-2-13C-1,3-15N2-hydantoin]. Quantification was achieved by an isotopic dilution technique: 5,5-di(pentadeuterophenyl)-hydantoin and 5-(4-hydroxy-3,5-dideuterophenyl)-5-phenyl-2-13C-1,3-15N2-hydantoin were used as internal standards. Molecular ion abundances of the permethylated derivatives were measured using a limited mass range repetitive scanning technique. The method is accurate, selective, reproducible and linear for analysis of 1.0 ml of serum and 0.5 ml of urine samples at the expected concentrations of drug (serum: 0.1-30.0 micrograms ml-1) and metabolite (serum: 0.1-10.0 micrograms ml-1; urine: 5.0-200.0 micrograms ml-1). The pharmacological equivalence of labeled and unlabeled phenytoin is demonstrated for a human volunteer. The results are discussed in the light of the further applications of the method, i.e. determination of the pharmacokinetics of a pulse dose of the labeled drug administered to patients who are taking a steady state dose of the unlabeled drug.
Animals↗
Application of thermospray liquid chromatography-mass spectrometry to the simultaneous quantification of tracer concentrations of isotopically labelled carbamazepine epoxide and steady-state levels of carbamazepine and carbamazepine epoxide.
A thermospray high-performance liquid chromatography-mass spectrometry method for the separation and quantification of tracer concentrations of isotopically labelled carbamazepine epoxide ([15N, 13C]CBZE) in the presence of steady-state levels of the anticonvulsant carbamazepine (CBZ) and its epoxide metabolite (CBZE) has been developed. The technique does not require derivatization, demonstrates little or no thermal degradation of the analytes, provides increased specificity not available from conventional high-performance liquid chromatography, and has a detection limit of 500 pg for CBZE on-column. The method, incorporating d4-CBZ and d4-CBZE as internal standards, allows precise and accurate determination of the analytes with good reproducibility and stability.
Carbamazepine↗
Practical aspects of uniform stable isotope labeling of higher plants for heteronuclear NMR-based metabolomics.
Analytical methods for probing plant metabolism are taking on new significance in the era of functional genomics, metabolomics, and systems biology. Nuclear magnetic resonance (NMR) is becoming a key technology in plant metabolomics. Stable isotope labeling of cultured cells and higher organisms has been especially promising in that it allows the use of advanced heteronuclear NMR methodologies through a combination of in vivo and in vitro measurements. This new approach provides much better resolution of the metabolite mixture signals in the multidimensional NMR spectra than does the conventional one-dimensional 1H-NMR previously used in plant metabolomics. In this chapter, we describe the practical aspects of two key NMR technologies: uniform stable labeling of plants and in vitro heteronuclear NMR.
Arabidopsis↗
Resonance Raman spectroscopy of the retinylidene chromophore in bacteriorhodopsin (bR570), bR560, M421, and other intermediates: structural conclusions based on kinetics, analogues, models, and isotopically labeled membranes.
Resonance Raman spectra of various intermediates in the bacteriorhodopsin proton pumping cycle have been obtained at physiological and low temperatures. To interpret these data, spectra of modél compounds, bacteriorhodopsin analogues, and isotopically labeled membranes have been measured. These results demonstrate that a protein group interacts with the Schiff base proton and, thus, the chromophore in protonated bacteriorhodopsin species is not a simple protonated Schiff base. This accounts for the abnormally low frequency of the C=N+H vibrational mode in bacteriorhodopsin and other failures to model the chromophore in bR570 with a simple butylamine protonated Schiff base of all-trans-retinal. To obtain the resonance Raman spectrum of M412 at physiological pH and temperatures, a dual beam kinetic technique was developed. We demonstrate that in the fingerprint region of the resonance Raman spectrum M412 is modeled accurately by a simple unprotonated butylamine Schiff base of all-trans-retinal. Spectral resolution and the solution environment of the membrane suspensions play important roles in this conclusion. Kinetic resonance Raman techniques are also used to monitor the time evolution of the M412 species and the intermediates which precede it. We find spectral features in our kinetic data which can be assigned to L550, and we present evidence for a new unprotonated species (X) which occurs before M412. Single pass flow resonance Raman spectra of bR560 also have been obtained, and, although bR570 and M412 appear to have all-trans chromophores, there are 13-cis-like features in the spectra of bR560, L550, and X.
Bacteriorhodopsins↗
Determination of verapamil in human plasma by mass fragmentography using stable isotope-labelled verapamil as internal standard.
In the present investigation a mass fragmentographic procedure for the quantitative determination of verapamil in human plasma was developed which makes use of the principle of mass spectrometry and of isotopic dilution: a known amount of isotopically labelled standard ([13C, 2H2]-verapamil) is added to the plasma sample. After an effective extraction procedure the ratio of the main fragments of verapamil (m/e 303) and the labelled standard (m/e 306) is measured by mass fragmentography. The lower limit of detection is at 1 ng/ml for plasma and at 10 pg/injection for pure verapamil. The precision was found to be between 3.4% and 14.4%, depending on the range (32.7 ng/ml and 2.2 ng/ml, resp.) of the concentration of verapamil in plasma.
Chemical Phenomena↗
Quantitative proteome analysis of CD95 (Fas/Apo-1)-induced apoptosis by stable isotope labeling with amino acids in cell culture, 2-DE and MALDI-MS.
Proteome analysis of Jurkat T cells induced to undergo apoptosis by CD95 (Fas/Apo-1) treatment was performed to identify modified proteins. We used stable isotope labeling with amino acids in cell culture (SILAC) using leucine to identify proteins of apoptotic and control Jurkat T cells by 2-DE and MALDI-MS. Out of 224 spots analyzed, we quantified 213 spots with 3.5 leucine-containing peptide pairs on average; 28 proteins with a relative abundance of higher than 1.5 were found. Five new modified proteins including calcyclin binding protein, cytosolic acyl coenzyme A thioester hydrolase, heterogeneous ribonucleoprotein M, replication factor C 37-kDa subunit, and tropomyosin 4 chain were identified as being modified in response to apoptosis. In comparison to differential proteome analysis via silver-stained 2-D gels and PMF of total Jurkat T cell lysates, 15 additional apoptosis-modified proteins were identified though 8 proteins were not found. The described approach using SILAC instead of silver staining for relative quantification was simpler to perform regarding the number of required 2-D gels, that cumbersome gel comparisons were avoided, and more apoptosis-modified proteins were identified, but with a higher demand on data interpretation of the mass spectra obtained.
Apoptosis↗
The use of stable isotope labeling and liquid chromatography-tandem mass spectrometry techniques to simultaneously determine the oral and ophthalmic bioavailability of timolol in dogs.
Assays have been established for the quantitation of timolol and its 13C3- and 2H9-stable-isotope-labeled analogs in plasma and urine using liquid chromatography with atmospheric-pressure chemical-ionization tandem mass spectrometry. For the analysis of urine, underivatized timolol and its labeled analogs are monitored while timolol in plasma is assayed down to concentrations of 0.2 ng/mL after derivatization with phosgene. The great power of this technique is illustrated by simultaneously assaying three different species of timolol in plasma and urine obtained from dogs receiving simultaneous ophthalmic, oral, and intravenous doses of unlabeled and [2H9]- and [13C3]-labeled timolol. Thus, the ophthalmic and oral bioavailabilities of timolol are measured in a single experiment rather than as a three-phase crossover experiment. This approach yields accurate and precise analytical data, obviates intrasubject variability, and saves both analytical and animal resources.
Administration, Oral↗
A dual-isotope-labeling method of studying the bioavailability of hexaglutamyl folic acid relative to that of monoglutamyl folic acid in humans by using multiple orally administered low doses.
BACKGROUND: The bioavailability of dietary folate may be hampered by the need of the glutamate moieties to be deconjugated before absorption. Previous studies comparing the bioavailabilities of polyglutamyl and monoglutamyl folic acid had inconsistent results. OBJECTIVE: The objective was to estimate the bioavailability of polyglutamyl relative to that of monoglutamyl folic acid by using a sensitive stable-isotope approach that allowed for the administration of multiple low doses in humans. DESIGN: Twenty subjects aged 20-50 y ingested 2 capsules daily for 28 d; each capsule contained approximately 50 nmol [(13)C(6)]hexaglutamyl and approximately 50 nmol [(13)C(11)]monoglutamyl folic acid. Amounts of the isotopically labeled compounds in the capsules were verified by various methods. The degrees of isotopic enrichment of plasma 5-methyltetrahydrofolate with (13)C(6) and (13)C(11) were measured by using liquid chromatography tandem mass spectrometry, and the ratio of (13)C(6) to (13)C(11) ((13)C(6):(13)C(11)) in plasma on day 28 was used as a measure of their relative bioavailability. RESULTS: The (13)C(11):(13)C(6) in plasma 5-methyltetrahydrofolate reached equilibrium on day 4 and was 0.66 (95% CI: 0.58, 0.74) on day 28. The (13)C(11):(13)C(6) content in the capsules varied between 1.18 and 1.96. After correction for this ratio, the estimated bioavailability of hexaglutamyl relative to that of monoglutamyl folic acid was >/=78%. CONCLUSION: Multiple dosing of low amounts of labeled folic acid is a sensitive, accurate, and efficient method of measuring the relative bioavailability of folic acid compounds, provided that the administered doses can be reliably assessed.
Administration, Oral↗
Acid hydrolysis of silk fibroins and determination of the enrichment of isotopically labeled amino acids using precolumn derivatization and high-performance liquid chromatography-electrospray ionization-mass spectrometry.
Silk fibroins from moth larvae and spiders are composed of highly repetitive Ala- and Gly-rich blocks that determine their structure, properties, and function. To investigate the metabolic integration of isotopically labeled amino acids in the excreted silk, the enrichment of ingested tracers was determined after acid hydrolysis of the fibroins. Thus, spiders and moth larvae were fed with stable isotope tracers such as [1-13C]Ala or [1-13C]Gly and silked. After hydrolysis of the silk proteins, the corresponding amino acids were derivatized with Nalpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent) and separated by liquid chromatography. The isotopical enrichment of the amino acids was determined by online electrospray mass spectrometry and calculated by newly developed software. Depending on the feeding protocol, enrichments of up to 58% in Gly and 31% in Ala were found in the investigated silks. The highly enriched silk fibroins are suitable for further structural investigation such as solid-state nuclear magnetic resonance.
Acetic Acid↗
Metal donation and apo-metalloenzyme activation by stable isotopically labeled metallothionein.
Coupled HPLC-ICP-MS has been used to quantitatively study the effects of GSSG and GSH on the ability of metallothionein (MTII) to donate essential and non-essential metals to apo-carbonic anhydrase. Stable isotopically labeled (67)Zn(3)Cd(4) MTII was used to enable Zn donated from MTII to be differentiated from extraneous sources of Zn. Transfer of both (67)Zn and Cd from MTII to apo-carbonic anhydrase was noted in the absence of either GSSG or GSH. GSSG increased the initial transfer of both Zn and Cd. Thereafter, a gradual increase in the (67)Zn content at the expense of Cd was noted over 24-h indicating continued interaction and exchange between MTII and the enzyme commensurate with the relative preferences shown by the proteins for these two metals. Although GSH also increased transfer of (67)Zn from MT it reduced the simultaneous transfer of Cd to the enzyme thereby conferring protection against Cd induced activation.
Cadmium↗
Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.
Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.
Cell Line↗
Tracking the in vivo fate of recombinant polypeptides by isotopic labeling.
We report a method to incorporate a stable isotope (13C) and a radioactive isotope (14C) into a recombinant polypeptide during Escherichia coli culture in M9 minimal medium supplemented with universally labeled 13C- or 14C-labeled glucose. We chose a thermally responsive elastin-like polypeptide (ELP) as a model polypeptide for this study because of its utility in various biotechnology applications such as drug delivery and tissue engineering. High cell densities were obtained by step-wise adaptation of E. coli to M9 medium in addition to supplementing the medium with trace elements that facilitated growth of E. coli. Furthermore, an optimal concentration of isopropyl-beta-d-thiogalactopyranoside was determined for induction of ELP expression to achieve high yield (mg/L culture) of the ELP. The incorporation of carbon isotopes was stoichiometrically related to the ratio of labeled glucose to unlabeled glucose in the culture medium. The isotope-labeled variants retained the physicochemical properties of the unlabeled ELP, specifically its temperature dependent aggregation behavior. As an example of the utility of this method, the in vitro stability of 14C-labeled ELP in PBS and mouse serum was conveniently quantified by SDS-PAGE and autoradiography. In addition, the in vivo stability of the 14C-labeled ELP in plasma was determined along with its plasma pharmacokinetics.
Autoradiography↗
Automated monitoring of phosphatidylcholine biosyntheses in Plasmodium falciparum by electrospray ionization mass spectrometry through stable isotope labeling experiments.
The metabolic pathways contributing to phosphatidylcholine biosyntheses in Plasmodium falciparum, the malaria-causing parasite, was explored by electrospray ionization mass spectrometry. Phosphatidylcholine produced by the CDP-choline pathway and by the methylation of phosphatidylethanolamine was identified and quantified through isotopic labeling experiments. A straightforward method based on cone voltage directed in-source fragmentations and relative abundance measurement of endogenous versus deuterated specific fragment ions was developed for simple and rapid automated data acquisition. Such high-throughput analytical protocol allowed us to measure the relative contribution of two different metabolic pathways leading to phosphatidylcholine without performing technically more demanding and time-consuming MS/MS or LC/MS experiments.
Animals↗
Biosynthesis of the pyrimidine moiety of thiamine independent of the PurF enzyme (Phosphoribosylpyrophosphate amidotransferase) in Salmonella typhimurium: incorporation of stable isotope-labeled glycine and formate.
Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-13C]glycine and [13C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.
Amidophosphoribosyltransferase↗
[Radioimmunodetection of human lung cancer xenografts by isotope labeling monoclonal antibody ALT-04 in nude mice].
Three nude mouse models bearing the human lung cancer were established from two fresh surgical specimens and one cell line. Tumor histological structure and cell morphology were similar before and after transplantation. The monoclonal antibody ALT-04 (McAb ALT-04) against human lung cancer was labeled by 125I, 131I and 201Tl. Radioimmunodetection study showed that all the three kinds of xenografts in nude mice were specifically located by McAb ALT-04. Imaging examination confirmed the ability of isotope labeling McAb ALT-04 to detect the presence of transplanted human lung cancer tissues without the aid of background subtraction manipulations. It is suggested that McAb ALT-04 have the possibility of locating in the tumor diagnosis and guiding in the treatment.
Animals↗
ATR-FTIR spectroscopy and isotope labeling of the PM intermediate of Paracoccus denitrificans cytochrome c oxidase.
The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).
Amino Acids↗