Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

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.     

Synthesis and application of 13C enriched phenylisothiocyanate as a 13C NMR protein probe

Phenylisothiocyanate, enriched with 13C at the isothiocyanate carbon, has been synthesized and utilized as a 13C NMR probe of proteins for the first time. The reagent has been used to label the amino groups of oxidized glutathione, and the resulting 13C NMR spectrum shows a prominent thiocarbonyl peak after a single NMR scan. The reagent is also capable of differentiating amino groups on the insulin molecule with distinct peaks corresponding to the amino groups on the A and B chains of insulin. This study illustrates the potential of using a new 13C label to functionalize amino groups of proteins and to study the labeled proteins with 13C NMR.     

Discrimination between12C and13C by marine plants

Summary The natural abundance¹³C/¹²C ratios (as δ¹³C) of organic matter of marine macroalgae from Fife and Angus (East Scotland) were measured for comparison with the species' ability to use CO₂ and HCO ₃ ^- for photosynthesis, as deduced from previously published pH-drift measurements. There was a clear difference in δ¹³C values for species able or unable to use HCO ₃ ^- . Six species of Chlorophyta, 12 species of Phaeophyta and 8 species of Rhodophyta that the pH-drift data suggested could use HCO ₃ ^- had δ¹³C values in the range -8.81‰ to -22.55‰. A further 6 species of Rhodophyta which the pH-drift data suggested could only use CO₂ had δ¹³C values in the range -29.90‰ to-34.51‰. One of these six species (Lomentaria articulata) is intertidal; the other five are subtidal and so have no access to atmospheric CO₂ to complicate the analysis. For these species, calculations based on the measured δ¹³C of the algae, the δ¹³C of CO₂ in seawater, and the known¹³C/¹²C discrimination of CO₂ diffusion and RUBISCO carboxylation suggest that only 15–21% of the limitation to photosynthesisin situ results from CO₂ diffusion from the bulk medium to the plastids; the remaining 79–85% is associated with carboxylation reactions (and, via feedback effects, down-stream processes). This analysis has been extended for one of these five species,Delesseria sanguinea, by incorporating data onin situ specific growth rates, respiratory rates measured in the laboratory, and applying Fick's law of diffusion to calculate a boundary layer thickness of 17–24 μm. This value is reasonable for aDelesseria sanguinea frondin situ. For HCO ₃ ^- -using marine macroalgae the range of δ¹³C values measured can be accommodated by a CO₂ efflux from algal cells which range from 0.306 of the gross HCO ₃ ^- influx forEnteromorpha intestinalis (δ¹³C=-8.81‰) in a rockpool to 0.787 forChondrus crispus (δ¹³C=-22.55‰). The relatively high computed CO₂ efflux for those HCO ₃ ^- -users with the more negative δ¹³C values implies a relatively high photon cost of C assimilation; the observed photon costs can be accommodated by assuming coupled, energy-independent inorganic carbon influx and efflux. The observed δ¹³C values are also interpreted in terms of water movement regimes and obtaining CO₂ from the atmosphere. Published δ¹³C values for freshwater macrophytes were compared with the ability of the species to use CO₂ and HCO ₃ ^- and again there was an apparent separation in δ¹³C values for these two groups. δ¹³C values obtained for marine macroalgae for which no pH-drift data are available permit predictions, as yet untested, as to whether they use predominantly CO₂ or HCO ₃ ^-     

Regulation of creatine phosphokinase B activity by protein kinase C

We previously reported that topical application of 12-o-tetradecanoylphorbol-13-acetate to mouse skin causes phosphorylation of epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34). In the accompanying paper, p40 was identified as creatine phosphokinase B. Here we report that both in intact cells and in a cell-free system, phosphorylation of creatine hosphokinase B by protein kinase C resulted in an increase in its ability to catalyze the transfer of the high-energy phosphate of phosphocreatine to ADP, thereby producing ATP. H-7, a specific inhibitor of protein kinase C was found to abolish the increase in enzyme activity. Lineweaver-Burk plot analysis indicated that the increased activity was mostly due to a decreased Km for phosphocreatine. Phosphorylation and activation of creatine phosphokinase B may be a physiological response to maintain ATP balance when a protein kinase C pathway is stimulated.     

cDNA cloning and mapping of the human creatine kinase M gene to 19q13

We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3.     

pH-dependence of 13C chemical shifts and 13C,H coupling constants in imidazole and L-histidine.

The pH-dependence of selected 13C chemical shifts reflects the state of ionization of the imidazole ring in both imidazole and L-histidine. Titration of the amino and carboxyl groups of histidine also perturbs the shifts. The coupling constants 1J (13C(2),H) and 1J (13C(5),H) for both compounds also vary with pH, but in L-histidine these constants are relatively insensitive to the titration of groups outside the imidazole ring.     

Metabolic response to [13C]glucose and [13C]fructose ingestion during exercise

Seven healthy male volunteers exercised on a cycle ergometer at 50 +/- 5% VO2max for 180 min, on three occasions during which they ingested either water only (W), [13C]glucose (G), or [13C]fructose (F) (140 +/- 12 g, diluted at 7% in water, and evenly distributed over the exercise period). Blood glucose concentration (in mM) significantly decreased during exercise with W (5.1 +/- 0.4 to 4.2 +/- 0.1) but remained stable with G (5.0 +/- 0.4 to 5.3 +/- 0.6) or F ingestion (5.4 +/- 0.5 to 5.1 +/- 0.4). Decreases in plasma insulin concentration (microU/ml) were greater (P less than 0.05) with W (11 +/- 3 to 3 +/- 1) and F (12 +/- 4 to 5 +/- 1) than with G ingestion (11 +/- 2 to 9 +/- 5), and fat utilization was greater with F (103 +/- 11 g) than with G ingestion (82 +/- 9 g) and lower than with W ingestion (132 +/- 14 g). However F was less readily available for combustion than G; over the 3-h period 75% (106 +/- 11 g) of ingested G was oxidized, compared with 56% (79 +/- 8 g) of ingested fructose. As a consequence, carbohydrate store utilizations were similar in the two conditions (G, 174 +/- 20 g; F, 173 +/- 17 g; vs. W, 193 +/- 22 g). These observations suggest that, during prolonged moderate exercise, F ingestion maintains blood glucose as well as G ingestion, and increases fat utilization when compared to G ingestion. However, due to a slower rate of utilization of F, carbohydrate store sparing is similar with G and F ingestions.     

13C NMR investigations on Npi-[13C1]carboxymethyl-histidine-119 ribonuclease.

The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12.     

Interaction of two prolamins with 1-13C oleic acid by 13C NMR

In this paper, we analyzed the interaction of Z19 prolamin from a BR451 maize variety and pennisetin from a BRS1501 pearl millet variety with 1-(13)C-enriched oleic acid (OA) by (13)C NMR in solution. In both proteins, we identified the presence of free fatty acids by NMR in solid state and solution. The interactions were analyzed at the protein/OA molar ratios of 1:1 and 1:4. In the Z19/OA 1:1 mixture in 70% ethanol and 30% D(2)O, the chemical shift of OA C1 was 182.9 ppm, about 3 ppm above that of the pure OA in the same solvent. In contrast, upon addition of OA to the pennisetin (1:1), the chemical-shift value slightly decreased by less than 1 ppm. The chemical-shift titration curve of OA C1 in an apparent pH range of 5.5-7.3 shifted by approximately 0.3 pH units toward higher pH values in the pennisetin/OA 1:1 complex relative to the pure OA. The results obtained for the pennisetin/OA 1:4 mixture were similar to the complexes at a 1:1 molar ratio. A significant difference was observed between the 1:1 and 1:4 curves for Z19. The titration curve for Z19/OA 1:1 suggested specific binding at the sites with electrostatic interaction.     

Muscle creatine uptake and creatine transporter expression in response to creatine supplementation and depletion.

The total creatine pool size [Cr(total); creatine (Cr) + phosphocreatine (PCr)] is crucial for optimal energy utilization in skeletal muscle, especially at the onset of exercise and during intense contractions. The Cr(total) likely is controlled by long-term modulation of Cr uptake via the sodium-dependent Cr transporter (CrT). To test this hypothesis, adult male Sprague-Dawley rats were fed 1% Cr, their muscle Cr(total) was reduced by approximately 85% [1% beta-guanidinoproprionic acid (beta-GPA)], or their muscle Cr(total) was repleted (1% Cr after beta-GPA depletion). Cr uptake was assessed by skeletal muscle (14)C-Cr accumulation to Cr and PCr by using hindlimb perfusion, and CrT protein content was assessed by Western blot. Cr uptake rate decreased with dietary Cr supplementation in the white gastrocnemius (WG; 45%) only. Depletion of muscle Cr(total) to approximately 15% of normal increased Cr uptake in the soleus (21%) and red gastrocnemius (22%), corresponding to 70-150% increases in muscle CrT content. In contrast, the inherently lower Cr uptake rate in the WG was unchanged with depletion of muscle Cr(total) even though CrT band density was increased by 230%. Thus there was no direct relationship between apparent muscle CrT abundance and Cr uptake rates. However, Cr uptake rates scaled inversely with decreases in muscle Cr(total) in the high-oxidative muscle types but not in the WG. This implies that factors controlling Cr uptake are different among fiber types. These observations may help explain the influence of initial muscle Cr(total), time dependency, and variations in muscle Cr(total) accumulation during Cr supplementation.     

13C-14C relations reveal that soil 13C-depth gradient is linked to historical changes in vegetation 13C

Plant and Soil - Plant-parasitic nematodes are able to sense and respond to gradients of chemical signals. How pH and inorganic salts in the rhizosphere affect nematode accumulation and...     

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.

     

Side chain dynamics monitored by 13C-13C cross-relaxation

A method to measure (13)C-(13)C cross-relaxation rates in a fully (13)C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves.The method has been used to measure (13)C-(13)C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on C(alpha)-C(beta) cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl (13)C-(13)C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The C(alpha)-C(beta) cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range C(beta)-C(delta) cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl C(delta) carbons is observed, which correlates well with the chi(2) torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion.