13C-NMR study of the binding of [1-13C]galactose-labelled N-acetyllactosamine and [1-13C]galactose-enriched hen ovalbumin to soybean agglutinin

The binding of the tide compounds to soybean agglutinin was investigated using 13C-NMR spectroscopy. The equilibrium constant for the binding of N-acetyllactosamine was found to be smaller than that obtained for the binding of ovalbumin (1.1 X 10(3) vs. 7.4 X 10(3) M-1). Only two binding sites per lectin tetramer were determined for the binding of ovalbumin, which is half the number of binding sites reported for the binding of small ligands to the lectin. Steric interference between the bulky ovalbumin molecules is believed to be the reason for the observed decrease in the apparent number of binding sites on the lectin.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

2D [1H,13C] NMR study of carbon fluxes during glucose utilization by Escherichia coli MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells--glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)--were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655(edd-eda), MG1655(zwf, edd-eda), and MG1655(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Metabolism of [2-13C]succinate in renal cells determined by 13C NMR

[2-13C]Succinate has been used to examine the metabolic carbon flux from the Krebs cycle in rat renal proximal convoluted tubular (PCT) cells under physiological and pathophysiological conditions. Therefore, we developed a mathematical model that enabled us to determine the metabolic fluxes of the Krebs cycle. A mathematical model for the calculation of flux from [2-13C]succinate was used to determine fluxes in rat PCT cells during chronic acidosis in the presence and absence of 0.1 mM angiotensin II. The relative carbon efflux via glutamate dehydrogenase in rat renal PCT cells increases during chronic acidosis from 0.27 to 0.39, whereas this carbon flux is not affected by the presence of peptide hormone angiotensin II in the incubation medium. The fraction of intermediate 13C-labelled oxaloacetate transformed into the phosphoenolpyruvate and aspartate pools increases significantly from 0.41 to 0.57 in the case of chronic acidosis. The carbon efflux is not affected by angiotensin II. The 13C-NMR data also show that the carbon efflux through phosphoenolpyruvate carboxykinase increases from 0.35 to 0.56 in rat renal PCT cells derived from chronic acidotic animals, as well as in the presence of angiotensin II. The present results indicate that angiotensin II affects only the flux through phosphoenolcarboxykinase, whereas chronic acidosis increases the flux through phosphoenolpyruvate carboxykinase as well as the gluconeogenic flux.     

Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy

• 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2-¹³C]acetate and ¹³C magnetic resonance spectroscopy.
• 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the ¹³C distribution in glucose where the overall ¹³C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean ¹³C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
• 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
• 4.4. With this carbon exchange factor and the approximated ¹³C enrichment of acetyl-CoA the intramitochondrial ¹³C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
• 5.5. Since acetate was metabolized solely in liver cells the ¹³C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
• 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
• 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the ¹³C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
• 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
• 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

     

1,2,3,4-13C] testosterone and [1,2,3,4-13C] estradiol

The preparation of [1,2,3,4-13C] testosterone and of [1,2,3,4-13C] estradiol by total synthesis is described. The 13C labels are introduced by alkylating intermediate 1 with [1,2,3,4-13C]l-iodo-3,3-ethylenedioxybutane (2) to obtain intermediate 10. Hydrolysis of the ketal function, cyclization, aromatization and removal of protective groups gave [1,2,3,4-13C] estradiol. Labeled testosterone was prepared by methylating intermediate 10 and by subsequent treatment with acid. The labeled steroids can be used as tracers for in vivo metabolic studies and as internal standards for the development of definitive gc-ms quantitative methods.     

Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR.

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.     

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

13C NMR studies on [4-13C] cholesterol incorporated in sonicated phosphatidylcholine vesicles

1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4-13C] cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. From the Dy3+ -induced shift of the C4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. At cholesterol concentrations below 30 mol percent the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.     

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

A (13)C NMR study on [3-(13)C]-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers: insertion, rigid-body motions, and local conformational fluctuations at ambient temperature

We have recorded (13)C NMR spectra of selectively [3-(13)C]Ala-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled synthetic transmembrane peptides of bacteriorhodopsin (bR) and enzymatically cleaved C-2 fragment in the solid and dimyristoylphosphatidylcholine bilayer. It turned out that these transmembrane peptides either in hexafluoroisopropanol or cast from it take an ordinary alpha-helix (alpha(I)-helix) irrespective of their amino acid sequences with reference to the conformation-dependent (13)C chemical shifts of (Ala)(n) taking the alpha-helix form. These transmembrane peptides are not always static in the lipid bilayer as in the solid state but undergo rigid-body motions with various frequencies as estimated from suppressed peaks either by fast isotropic or large-amplitude motions (>10(8) Hz) or intermediate frequencies (10(5) or 10(3) Hz). Further, (13)C chemical shifts of the [3-(13)C]Ala-labeled peptides in the bilayer were displaced downfield by 0.3-1.1 ppm depending upon amino acid sequence with respect to those in the solid state, which were explained in terms of local conformational fluctuation (10(2) Hz) deviated from the torsion angles (alpha(II)-helix) from those of standard alpha-helix, under anisotropic environment in lipid bilayer, in addition to the above-mentioned rigid-body motions. The carbonyl (13)C peaks, on the other hand, are not sensitively displaced by such local anisotropic fluctuations, because they are more sensitive to the manner of hydrogen-bond interactions. The amino acid sequences of these peptides inserted within the bilayer were not always the same as those of intact bR, causing disposition of the transmembrane alpha-helical segment from that of intact bR. Finally, we confirmed that the (13)C NMR peak positions of the random coil form are located at the boundary between the alpha-helix and a turned structure in loop regions.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

Carbon-13 NMR studies on [4-13C] uracil labelled E. coli transfer RNA1(Val1).

In this paper we describe carbon-13 nuclear magnetic resonance results on 13C-enriched purified transfer RNAI(VAL) from from E. coli SO-187, a uracil requiring auxotroph. The organism was grown on uracil 90% 13C-enriched at the carbonyl C4 position. Transfer RNAI(Val) was purified from bulk tRNA by sequential chromatography on columns of BD cellulose, DEAE-Sephadex A-50 and reverse gradient sepharose 4B. Dihydrouridine, 4-thiouridine, and uridine 5-oxyacetic acid located at discrete positions in the polymer backbone were tentatively assigned in the highly resolved 25 MHz 13C-spectra. Chemical shift versus temperature plots reveal differential thermal perturbation of the ordered solution structure, evident in the large dispersion (ca 3-4 ppm) of the uridine C4 resonances. Over the range 26-68 degrees C, V in the anticodon displays the largest downfield shift. Whereas several uridine residues rapidly shift downfield between 50-68 degrees, one moves upfield beginning at 37 degrees. The results are qualitatively compared with proton NMR analysis of the three dimensional structure.     

NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].