Relative concentrations of N15 in urinary ammonia N and urea N after feeding N15-labeled compounds

Available data on the isotopic ratio See PDF for Equation of ammonia (r_a) and that of urea (r_u) after a single feeding of glycine, aspartic acid, and ammonium citrate are analyzed. From this analysis the following conclusions are drawn. 1. The isotopic ratio See PDF for Equation of ammonia (r_a) is always higher than that of urea (r_u) in the initial period after a single feeding of isotopic glycine or aspartic acid, but the relation is reversed later. A similar relation probably holds after feeding isotopic ammonia. 2. It is pointed out that the ratio of average r_a to average r_u depends on the time interval for which urine is collected, on the schedule of feeding, and probably also on the amount taken at each feeding. When the amount fed and the feeding schedule are unknown, theoretical interpretation of the ratio of average r_u to average r_u is impossible. 3. At the point of maximum isotopic ratio of urea, it is very probably equal to the isotopic ratio of ammonia. A possible explanation is suggested.     

Transverse relaxation optimized 3D and 4D 15N/15N separated NOESY experiments of 15N labeled proteins

Distance constraints are an important complement to orientational constraints. While a high-resolution monomer structure of the ion channel forming polypeptide, gramicidin A, has been solved with 120 orientational constraints, the precise geometry of the dimer interface has not been characterized. Here, using both ¹³C and ¹⁵N labeled gramicidin A samples in hydrated phospholipid bilayers, both inter- and intramolecular distances have been measured with a recently developed simultaneous frequency and amplitude modulation (SFAM) solid-state NMR scheme. Using this approach ¹⁵N-¹³C₁ residual dipolar couplings across a hydrogen bond as small as 20 ± 2 Hz have been characterized. While such distances are on the order of 4.2 ± 0.2 Å, the spectroscopy is complicated by rapid global motion of the molecular structure about the bilayer normal and channel axis. Consequently, the nominal 40 Hz dipolar coupling is averaged depending on the orientation of the internuclear vector with respect to the motional axis. The intermolecular distance confirmed the previously described monomeric structure, while the intramolecular distance across the monomer–monomer interface defined this junction and confirmed the previous model of this interface.     

Rate of excretion of N15 after feeding N15-labeled l-aspartic acid in man

1. l-Aspartic acid labeled with N¹⁵ was fed to one human adult and six infants, and the total N and N¹⁵ were determined in the urine from time to time. 2. The N¹⁵ concentration (or isotopic ratio) of urinary N reached its maximum in the adult about 2 hours and in the infants about 4 hours after feeding, then fell off logarithmically. 3. Assuming that the N of aspartic acid readily entered into equilibrium with other N compounds in the pool, the rate of turnover of the N pool was calculated from the rate of fall of the isotopic ratio of urinary N. This rate of turnover of N was about 4 per cent per hour in the adult and 6 to 12 per cent per hour in the infants. 4. The rate of protein synthesis calculated from the rate of turnover of N was 10 mg. N per kilo per hour in the adult and 18 to 27 mg. N per kilo per hour in the infants, with one exception which showed a higher rate of 52 mg. N per kilo per hour. The size of the metabolic pool of N per kilo in non-growing infants was about the same as that in the adult (0.4 to 0.5 gm.) but it was somewhat larger in growing infants (0.5 to 0.8 gm.).     

Box-modeling of 15N/14N in mammals

The ¹⁵N/¹⁴N signature of animal proteins is now commonly used to understand their physiology and quantify the flows of nutrient in trophic webs. These studies assume that animals are predictably ¹⁵N-enriched relative to their food, but the isotopic mechanism which accounts for this enrichment remains unknown. We developed a box model of the nitrogen isotope cycle in mammals in order to predict the ¹⁵N/¹⁴N ratios of body reservoirs as a function of time, N intake and body mass. Results of modeling show that a combination of kinetic isotope fractionation during the N transfer between amines and equilibrium fractionation related to the reversible conversion of N-amine into ammonia is required to account for the well-established ≈4‰ ¹⁵N-enrichment of body proteins relative to the diet. This isotopic enrichment observed in proteins is due to the partial recycling of ¹⁵N-enriched urea and the urinary excretion of a fraction of the strongly ¹⁵N-depleted ammonia reservoir. For a given body mass and diet δ¹⁵N, the isotopic compositions are mainly controlled by the N intake. Increase of the urea turnover combined with a decrease of the N intake lead to calculate a δ¹⁵N increase of the proteins, in agreement with the observed increase of collagen δ¹⁵N of herbivorous animals with aridity. We further show that the low δ¹⁵N collagen values of cave bears cannot be attributed to the dormancy periods as it is commonly thought, but inversely to the hyperphagia behavior. This model highlights the need for experimental investigations performed with large mammals in order to improve our understanding of natural variations of δ¹⁵N collagen.     

Incorporation of 15N into allantoin in nodulated soybean plants supplied with 15N2

Incorporation of ¹⁵N into allantoin and allantoic acid in noduleswas higher than that in roots. This confirms that nodules produceallantoin. The ¹⁵N concentration in allantoin was slightly higherthan that in allantoic acid, suggesting that allantoin decomposedto allantoic acid. Allantoin and allantoic acid in nodules weretranslocated rapidly to roots. (Received August 25, 1976; )     

Solid-state 15N NMR analysis of highly 15N-enriched plant materials

Solid-state ¹⁵N NMR spectroscopy was used to determine the chemical nature of nitrogen in ¹⁵N-enriched material from the roots and stems of wheat (Triticum aesitivum), field pea (Pisum sativum) and kikuyu grass (Pennisetum clandestinum) and from the roots, stems and leaves of a eucalyptus species (Eucalyptus globulus). Nitrogen-15 cross polarization (CP) spectra of the materials were all very similar, with 64–75% of total signal assigned to amide N. Spin counting analysis indicated that 37–80% of potential signal was accounted for in the CP spectra, and that NMR observability using the CP technique (N _obs -CP) was higher for stems and leaves than for roots, and higher for wheat and eucalyptus than for peas and kikuyu. The ¹⁵N direct polarization (DP) spectra contained higher proportions of signal assigned to amine (up to 22%) and nitrate (up to 17%), and less assigned to amide N (50–72%) than the corresponding CP spectra. Spin counting analysis indicated that 68–93% of potential signal was accounted for in the DP spectra, confirming the DP technique to be more quantitatively reliable than CP.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

N15: the linear phage-plasmid

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.     

15N natural abundances and N use by tundra plants

Plant species collected from tundra ecosystems located along a north-south transect from central Alaska to the north coast of Alaska showed large and consistent differences in ¹⁵N natural abundances. Foliar ¹⁵N values varied by about 10% among species within each of two moist tussock tundra sites. Differences in ¹⁵N contents among species or plant groups were consistent across moist tussock tundra at several other sites and across five other tundra types at a single site. Ericaceous species had the lowest ¹⁵N values, ranging between about –8 to –6. Foliar ¹⁵N contents increased progressively in birch, willows and sedges to maximum ¹⁵N values of about +2 in sedges. Soil ¹⁵N contents in tundra ecosystems at our two most intensively studied sites increased with depth and ¹⁵N values were usually higher for soils than for plants. Isotopic fractionations during soil N transformations and possibly during plant N uptake could lead to observed differences in ¹⁵N contents among plant species and between plants and soils. Patterns of variation in ¹⁵N content among species indicate that tundra plants acquire nitrogen in extremely nutrient-poor environments by competitive partitioning of the overall N pool. Differences in plant N sources, rooting depth, mycorrhizal associations, forms of N taken up, and other factors controlling plant N uptake are possible causes of variations in ¹⁵N values of tundra plant species.     

Synthesis and 15N NMR of d[CGT(15N6)ACG] and d[CGT(CGT)(15N1)ACG]

Abstract

Deoxyadenosine has been converted to 6.¹⁵N deoxyadenosine, which in turn has been transformed to 1-¹⁵N deoxyadenosine. Each of these ¹⁵N derivatives was then incorporated into the hexanucleoside pentaphosphate d(CGTACG) via a phosphoramidite procedure. The monomers and die hexamers were characterized by ¹H and ¹⁵N nmr.     

Cross-peptide bond 13C--15N coupling constants by 13C and J cross-polarization 15N NMR

Comparative 13C--15N coupling constants are reported for the linear dipeptide tBoc-L-[U-13C]Ala-[15N]GlyOMe and the corresponding cyclic diketopiperazine, both in dimethylsulfoxide (DMSO) and, upon removal of the tBoc group, in water solutions. Spectra were obtained by 13C NMR and by the first application of J cross-polarization (JCP) 15N NMR, which greatly reduces the time required to accumulate 15N NMR spectra. In DMSO there was evidence for the formation of complexed species which were not present in water. The values obtained for the cross-peptide bond coupling constant 2J13C alpha--15N were consistently less (by 2.2 Hz in DMSO, 4.3 Hz in water) for the cyclic than for the linear peptide, which may be related to the cross-peptide bond conformation. The 15N resonance for the cyclic peptide was shifted only 2 ppm downfield from the linear peptide chemical shift value in both solvents.