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PubMed2001

Stable isotope-labeling studies on the oxidative coupling of caffeic acid via o-quinone.

The formation of ortho-quinone from ortho-diphenol is a key step in its dimerization. An NMR analysis of the oxidation of 3,4-dihydroxycinnamic acid (caffeic acid) by NaIO4 revealed the formation of 3-(3',4'-dioxo-1',5'-cyclohexadienyl) propenoic acid (o-quinone) prior to the formation of furofuran-type lignan 4,8-exo-bis (3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione. Both electrolytic and enzymatic oxidation of caffeic acid also generated o-quinone. The yields of o-quinone from caffeic acid were quantified by NMR and HPLC analyses. A stable isotope-labeling study of the formation of lignans directly proved the random radical coupling of semiquinone radicals formed from a set of caffeic acid and o-quinone.

Benzoquinones↗
PubMed2004

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.

The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.

Chromatography↗
PubMed2000

Simulations of oligopeptide vibrational CD: effects of isotopic labeling.

Simulated ir absorption and vibrational CD (VCD) spectra of four alanine-based octapeptides, each having its main chain constrained to a different secondary structure conformation, were analyzed and compared with experimental results for several different peptides. The octapeptide simulations were based on transfer of property tensors from a series of ab initio calculations for a short L-alanine based segment containing 3 peptide bonds with relative straight phi, psi angles fixed to those appropriate for alpha-helix, 3(10)-helix, ProII-like helix, and beta-sheet-like strand. The tripeptide force field (FF) and atomic polar tensors were obtained with density functional theory techniques at the BPW91/6-31G** level and the atomic axial tensor at the mixed BPW91/6-31G**/HF/6-31G level. Allowing for frequency correction due to the FF limitations, the octapeptide results obtained are qualitatively consistent with experimental observations for ir and VCD spectra of polypeptides and oligopeptides in established conformations. In all cases, the correct VCD sign patterns for the amide I and II bands were predicted, but the intensities did have some variation from the experimental patterns. Predicted VCD changes upon deuteration of either the peptide or side-chains as well as for (13)C isotopic labeling of the amide C=O at specific sites in the peptide chain were computed for analysis of experimental observations. A combination of theoretical modeling with experimental data for labeled compounds leads both to enhanced resolution of component transitions and added conformational applicability of the VCD spectra.

Alanine↗
PubMed1997

Identification of modification sites in large biomolecules by stable isotope labeling and tandem high resolution mass spectrometry. The active site nucleophile of thiaminase I.

A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (Mr) has been increased appropriately by the modification. As found previously for MS of small molecules, this study shows that protein fragment identification can be greatly simplified by labeling the modification with stable isotopes. Further, the high resolution capabilities of Fourier transform MS make possible the direct identification of CH3/CD3-labeled peptides without chromatographic separation. Although separate Asp-N, Lys-C, and alpha-chymotrypsin digests of thiaminase I (42 kDa) yielded as many as 70 peptides, FTMS identification of the labeled peptide localized the modification site of a mechanism-based inhibitor to Arg101-Lys121, Asp90-Gly122, and Gly107-Tyr119, respectively. The measured mass difference values of the two labels agreed with that expected for CH3/CD3, 3.019 Da, with a standard deviation of 0.005 Da, providing persuasive identity verification. MS/MS fragmentation narrowed the site to Pro109-Phe118 and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys113 as the thiaminase I active-site nucleophile among the 379 amino acids.

Alkyl and Aryl Transferases↗
PubMed1983

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. II. A correction.

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Isotopic labeling and EPR spectroscopy show that a tyrosine residue is the terminal electron donor, Z, in manganese-depleted photosystem II preparations.

Photosystem II contains two redox active species, D and Z, which in the oxidized and paramagnetic form give rise to similar EPR signals. Both signals are broadened by anisotropic interactions and show no well-resolved hyperfine splittings, even at room temperature. D+ is a dark stable radical of unknown function. Z, on the other hand, acts as the intermediate electron carrier between the primary chlorophyll donor and the manganese catalytic site. In manganese-depleted photosystem II preparations, where Z+ is the terminal electron donor, the Z+ EPR signal can be easily detected. Isotopic labeling of intact cells of Synechocystis 6803 and EPR spectroscopy were used to demonstrate that D+ is a tyrosine radical (Barry, B.A., and Babcock, G. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7099-7103). However, since the Z+ signal is not observable in intact cyanobacterial cells, a definitive assignment of the Z redox active species has not yet been performed. Here, we describe the use of a purified photosystem II preparation to assign the Z+ EPR signal to a neutral tyrosine radical. Further, a comparison of the Z+ and D+ EPR lineshapes in deuterated samples shows that there are slight differences between the two signals. These alterations may be caused by differences in the protein environments of the D+ and Z+ radicals.

Cyanobacteria↗
PubMed1983

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations.

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.

Cell Separation↗
PubMed2004

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry.

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.

Amino Acid Sequence↗
PubMed1980

Release of radio-isotope labelled antigens from Plasmodium knowlesi during merozoite re-invasion in vitro.

Labelled antigens sharing determinants with both membrane and cytoplasmic fractions of Plasmodium knowlesi were detected in culture medium after in vitro incubation of schizont-stage parasites previously pulse-labelled with [3H]isoleucine. Release of antigens occurred only during schizont rupture and merozoite re-invasion. Antigenic material accounted for up to a third of the total trichloroacetic acid-insoluble radioactivity released by the cells. Absorption experiments indicated that approximately two-thirds of this antibody-precipitable material shared determinants with parasite membrane antigens, with a similar quantity sharing determinants with cytoplasmic proteins. Using antisera derived against 2 different antigenic variants of P. knowlesi, no evidence for the release of variant-spcific antigens was obtained. Centrifugal analysis revealed that the majority of the radio-isotope labelled antigens were particulate in nature; however, some appeared to exist in true solution.

Animals↗
PubMed2004

The in vivo use of the stable isotope-labelled biomarkers lactose-[15N]ureide and [2H4]tyrosine to assess the effects of pro- and prebiotics on the intestinal flora of healthy human volunteers.

Amongst the various claimed beneficial effects of pro- and prebiotics for the human host, it has been hypothesised that functional foods are able to suppress the generation and accumulation of toxic fermentation metabolites (NH3, p-cresol). Direct evidence supporting this hypothesis is lacking mainly because of the unavailability of reliable biomarkers. Preliminary data indicate that lactose-[15N]ureide and [2H4]tyrosine may be potential biomarker candidates. The aim of the present study was to evaluate the effect of pro- and prebiotics on the colonic fate of these biomarkers in a randomised, placebo-controlled, cross-over study with nineteen healthy volunteers. At the start of the study and at the end of each 2-week study period, during which they were administered either a probiotic (n 10; 6.5 x 10(9) Lactobacillus casei Shirota cells twice daily) or a prebiotic (n 9; lactulose 10 g twice daily), the volunteers consumed a test meal containing the two biomarkers. Urine was collected during 48 h. Results were expressed as percentage of the administered dose. As compared with the placebo, the decrease in the percentage dose of p-[2H4]cresol in the 24-48 h urine fraction was significantly higher after probiotic intake (P=0.042). Similar changes were observed for the 15N tracer (P=0.016). After prebiotic intake, a significantly higher decrease in the percentage dose of p-[2H4]cresol (P=0.005) and 15N tracer (P=0.029) was found in the 0-24 h urine collection. The present results demonstrate that suppression of the generation and accumulation of potentially toxic fermentation metabolites by pro- and prebiotics can reliably be monitored in vivo by the use of stable isotope-labelled biomarkers.

Adult↗
PubMed1996

The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation.

The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.

Amino Acid Sequence↗
PubMed2001

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation.

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

Algorithms↗
PubMed1983

Separation of unlabeled and isotopically-labeled metabolites of arachidonic acid by argentation high-pressure liquid chromatography.

Argentation HPLC can be used to separate unlabeled metabolites of 20:4 from their deuterium and tritium-labeled analogs. In many cases baseline resolution of labeled and unlabeled forms is achieved. This method can be used to obtain labeled compounds of high isotopic purity which are suitable for use as standards in radioimmunoassay or GC-MS using selected ion monitoring.

Animals↗
PubMed1986

Resonance Raman studies of isotopically labeled chloroperoxidase.

Chloroperoxidase (CPO) and cytochrome P450cam have been shown by several techniques to have similar active site properties. Recent resonance Raman investigations using isotopically enriched 34S-labeled samples have demonstrated thiolate ligation in the P450cam system. We report here on a number of parallel studies involving CPO. On the basis of isotopic labeling (34S, 13CO), we assign the Fe-S and Fe-CO stretching frequencies of CPO at 347 (-vFe-S) and 488 cm-1 (-vFe-CO). The differences of the -vFe-S and -vFe-CO in CPO and P450cam may suggest subtle differences in the thiolate binding in the two systems.

Chloride Peroxidase↗