NMR structure analysis of uniformly 13C-labeled carbohydrates

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of ¹³C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly ¹³C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-¹³C)-sucrose, 342 Da] and one compound of medium molecular weight (¹³C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The ¹³C resonances are traced using ¹³C–¹³C correlations from homonuclear experiments, such as (H)CC–CT–COSY, (H)CC–NOESY, CC–CT–TOCSY and/or virtually decoupled (H)CC–TOCSY. Based on the assignment of the ¹³C resonances, the ¹H chemical shifts are derived in a straightforward manner using one-bond ¹H–¹³C correlations from heteronuclear experiments (HC–CT–HSQC). In order to avoid the ¹ J _CC splitting of the ¹³C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either ¹³C or ¹H detected experiments, namely CC–CT–COSY, band-selective (H)CC–TOCSY, HC–CT–HSQC–NOESY or long-range HC–CT–HSQC. However, due to the short T₂ relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the ¹H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the ¹³C-enriched polysaccharide were assigned by using HC–H2BC spectra. The assignment of the N-acetyl groups with ¹⁵N at natural abundance was completed by using HN–SOFAST–HMQC, HNCA, HNCO and ¹³C-detected (H)CACO spectra.     

Retrobiosynthetic nuclear magnetic resonance analysis of amino acid biosynthesis and intermediary metabolism. Metabolic flux in developing maize kernels

Information on metabolic networks could provide the basis for the design of targets for metabolic engineering. To study metabolic flux in cereals, developing maize (Zea mays) kernels were grown in sterile culture on medium containing [U-(13)C(6)]glucose or [1,2-(13)C(2)]acetate. After growth, amino acids, lipids, and sitosterol were isolated from kernels as well as from the cobs, and their (13)C isotopomer compositions were determined by quantitative nuclear magnetic resonance spectroscopy. The highly specific labeling patterns were used to analyze the metabolic pathways leading to amino acids and the triterpene on a quantitative basis. The data show that serine is generated from phosphoglycerate, as well as from glycine. Lysine is formed entirely via the diaminopimelate pathway and sitosterol is synthesized entirely via the mevalonate route. The labeling data of amino acids and sitosterol were used to reconstruct the labeling patterns of key metabolic intermediates (e.g. acetyl-coenzyme A, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and Rib 5-phosphate) that revealed quantitative information about carbon flux in the intermediary metabolism of developing maize kernels. Exogenous acetate served as an efficient precursor of sitosterol, as well as of amino acids of the aspartate and glutamate family; in comparison, metabolites formed in the plastidic compartments showed low acetate incorporation.     

Quantitative radiochromatographic analysis of the major groups of carbohydrates in cultured animal cells.

Procedures are described for selective quantitation of the monosaccharide content of glycogen, chondroitin sulfates, hyaluronic acid, glycoproteins, glycolipids, N-acetylneuraminic acid, and the phosphorylated carbohydrate pools in cultured animal cells. Monosaccharides are released from each type of carbohydrate by selective hydrolysis with enzymes and/or acid and are analyzed by radiochromatographic procedures which give reliable quantitative data with only a few nanomoles of each monosaccharide. Analyses of the entire spectrum of carbohydrates can be carried out using 7–8 mg of animal cell protein.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Metabolism of methanol in plant cells. Carbon-13 nuclear magnetic resonance studies

Using (13)C-NMR, we demonstrate that [(13)C]methanol readily entered sycamore (Acer pseudoplatanus L.) cells to be slowly metabolized to [3-(13)C]serine, [(13)CH(3)]methionine, and [(13)CH(3)]phosphatidylcholine. We conclude that the assimilation of [(13)C]methanol occurs through the formation of (13)CH(3)H(4)Pte-glutamate (Glu)(n) and S-adenosyl-methionine, because feeding plant cells with [3-(13)CH(3)]serine, the direct precursor of (13)CH(2)H(4)Pte-Glu(n), can perfectly mimic [(13)CH(3)]methanol for folate-mediated single-carbon metabolism. On the other hand, the metabolism of [(13)C]methanol in plant cells revealed assimilation of label into a new cellular product that was identified as [(13)CH(3)]methyl-beta-D-glucopyranoside. The de novo synthesis of methyl-beta-D-glucopyranoside induced by methanol did not require the formation of (13)CH(3)H(4)Pte-Glu(n) and was very likely catalyzed by a "transglycosylation" process.     

Enhancement of norepinephrine biosynthesis by ascorbic acid in cultured bovine chromaffin cells

Ascorbic acid donates electrons to dopamine beta-monooxygenase during the hydroxylation of dopamine to norepinephrine in vitro. However, the possible role of ascorbic acid in norepinephrine biosynthesis in vivo has not been defined. We therefore investigated the effect of newly accumulated ascorbic acid on catecholamine biosynthesis in cultured bovine adrenal chromaffin cells. Cells supplemented for 3 h with ascorbic acid accumulated 9-fold more ascorbic acid than found in control cells. Under these conditions, the cells loaded with ascorbate were found to double the rate of norepinephrine biosynthesis from [14C]tyrosine compared to control. By contrast, the amounts present of [14C] 3,4-dihydroxyphenylalanine and [14C]dopamine synthesized from [14C]tyrosine were unaffected by the preloading of ascorbic acid. Ascorbate preloaded cells incubated with [3H]dopamine also showed a similar increase in the rate of norepinephrine formation, without any change in dopamine transport into the cells. Thus, these data were consistent with ascorbate action at the dopamine beta-monooxygenase step. In order to determine if ascorbate could interact directly with dopamine beta-monooxygenase localized within chromaffin granules, we studied whether isolated chromaffin granules could accumulate ascorbic acid. Ascorbic acid was not transported into chromaffin granules by an uptake or exchange process, despite coincident [3H]dopamine uptake which was Mg-ATP dependent. These data indicate that ascorbic acid does augment norepinephrine biosynthesis in intact chromaffin cells, but by a mechanism that might enhance the rate of dopamine hydroxylation indirectly.     

Microinjections of cultured rat conceptuses: studies with 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide.

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

A constitutional analysis of some 14C-labeled carbohydrates of high specific activity by carbon-13 n.m.r. spectroscopy and field-desorption,mass spectrometry

The conventional methods of determining the labeling pattern in organic molecules are very laborious and time-consuming, because they rely on carbon-by-carbon chemical degradations. The determination of labeling patterns in carbohydrates by means of ¹³C-n.m.r. spectroscopy is now described for the first time. A rapid (30 min), micro (?I μg) method for determining the distribution of various isotopomers (which can be used to calculate specific activity), using f.d.m.s., is also described.     

The biosynthesis of tabtoxinine-beta-lactam. Use of specifically 13C-labeled glucose and 13C NMR spectroscopy to identify its biosynthetic precursors

Tabtoxinine-beta-lactam, an irreversible inhibitor of glutamine synthetase is produced by several pathovars of Pseudomonas syringae. We have examined tabtoxinine-beta-lactam biosynthesis, an important and poorly characterized step in pathogenesis caused by this organism. We have identified the biosynthetic precursors of tabtoxinine-beta-lactam by incorporating 13C from specifically 13C-labeled D-glucose precursors and determining the labeling pattern using 13C NMR spectroscopy. Tabtoxinine-beta-lactam is generated by combining a 4-carbon fragment, a 2-carbon fragment, and a single carbon. The 4-carbon fragment arises from aspartic acid, and the 2-carbon unit is donated from carbons 2 and 3 of pyruvate. The 6-carbon backbone of tabtoxinine-beta-lactam arises from the condensation of fragments from aspartate and pyruvate, probably using reactions analogous to the initial steps in the pathway of lysine biosynthesis.     

Synthesis of (13)C-labeled gamma-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77K induced the EPR-spectrum of a neutral flavin semiquinone (g=2.005, linewidth 2.1 mT), while at 10K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with (13)C have been synthesized. This was achieved via (13)C-labeled gamma-butyrolactones, which were obtained from (13)C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the (13)C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.     

Microinjection of cultured rat embryos: studies with retinol, 13-cis- and all-trans-retinoic acid

Retinol, all-trans-retinoic acid or 13-cis-retinoic acid were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships of the retinoids showed that the dysmorphogenic effects were qualitatively similar for all three, but were elicited by a low concentration of all-trans-retinoic acid (250 ng/ml), a 6- to 7-fold higher concentration of 13-cis-retinoic acid and an approximately 16-fold higher concentration of retinol. After microinjection of 2,000 ng/ml of retinol, no dysmorphogenesis was observed but instead an increase in all growth parameters as compared to the controls.