Biosynthetic preparation of cardenolides from [1-14C]acetic acid by stem discs of the milkweed Asclepias curassavica

[¹⁴C]Calotropin (11.2 μCi/mmol) and uscharidin (14.1 μCi/mmol) were biosynthesized by stem discs of Asclepias curassavica incubated in a medium containing [1-¹⁴C]acetic acid. Relative isotope enrichment sites determined by ¹³C NMR spectroscopy of [¹³C]calotropin prepared by the same method were at C-23 (0.71 %), C-2′ (0.28 %) and C-4′ (0.21 %).     

13C-NMR studies on griseofulvin biosynthesis and acetate metabolism in Penicillium patulum

Incorporations of singly and doubly-labelled acetate-[¹³C] into griseofulvin by a mutant strain of Penicillium patulum confirm its origin from simple folding of a single heptaketide chain. An acetate ‘starter’ effect is observed in the ¹³C-NMR spectra of griseofulvin enriched from acetate-[¹³C], and analysis of the ¹³C—¹³C spin—spin couplings observed indicate a rapid metabolic turnover of added acetate. Methyl, but not carboxyl, of acetate is efficiently metabolised into the C₁ pool.     

An NMR study of the loss of carbon-20 in the biosynthesis of gibberellin A3 by Gibberella fujikuroi

Resuspension cultures of Gibberella fujikuroi, strain GF-1a, were shown to metabolise potassium [3′-¹³C] mevalonate to ¹³C-enriched C₁₉-gibberellins, plus ¹³CO₂ (derived from the loss of carbon-20). The formation of [¹³C]-gibberellins could be observed in vivo using ¹³C NMR; however that of ¹³CO₂ could not. In contrast, removal of the mycelium and concentration of the filtrate at pH 12 enabled the ¹³CO₂ produced to be observed using ¹³C NMR. During incubations of H¹⁴CO₂Na with this fungus, complete conversion to other radioactive products was observed, and the significance of these results in the light of previous work is discussed.     

An investigation of the mechanisms for sterol synthesis and dietary sterol bioconversion in the heterotrophic protists Oxyrrhis marina and Gyrodinium dominans

The ability of the marine heterotrophic protists Oxyrrhis marina and Gyrodinium dominans to synthesize sterols de novo and modify dietary sterols was investigated using ¹³C-labeled substrates. De novo sterol synthesis of O. marina was determined by incorporation of ¹³C acetate into the culture medium. For G. dominans which has low tolerance of acetate, a protozoan prey Perkinsus marinus that cannot synthesize sterols, was cultured with ¹³C acetate then fed to G. dominans. Both heterotrophs utilized dietary ¹³C to synthesize fatty acids de novo, but not sterols. The ability of O. marina and G. dominans to alkylate, saturate, and desaturate dietary sterols was tested using P. marinus incorporated with ¹³C-labeled cholesterol as prey. O. marina did not modify the dietary ¹³C-cholesterol, but G. dominans produced 5 labeled sterols (brassicasterol, C28:1, and unknown C28, C29 and C30 sterols) indicating that G. dominans has the ability to desaturate and alkylate dietary cholesterol. The ability of O. marina and G. dominans to dealkylate dietary sterols was tested by feeding them gelatin acacia microspheres (GAMs) containing ¹³C-labeled brassicasterol. Neither heterotroph dealkylated brassicasterol to make cholesterol, but G. dominans alkylated and saturated brassicasterol to make 2 sterols (C29:1 and C30:0). The lack of dealkylation of brassicasterol by both protist species suggests problems with the substrate and/or delivery system since previous studies suggest that dealkylation of brassicasterol occurs when either species is fed algae containing this sterol.     

Changes in the oceanic 13C/12C ratio during the last 140 000 years: High-latitude surface water records

Detailed records of the carbon and oxygen isotopic ratios of Neogloboquadrina pachyderma are compared between nine high-latitude sediment cores, from the Northern and Southern Hemispheres, covering the last 140 000 yrs. The strong analogies between the δ¹³C records permit to define a δ¹³C stratigraphic scale, with three clear cut transitions simultaneous with the oxygen isotopic transitions 6/5 (125 kyrs.), 5/4 (65 kyrs.), and 2/1 (13 kyrs.). The δ¹³C records of N. pachyderma in the high-latitude cores, which follow the changes in δ¹³C of the surface water TCO₂ near areas of deep water formation present trends similar to the benthic foraminifera δ¹³C records in cores V19–30 and M12-392, although amplitudes of the isotopic shifts are different. This implies that a large part of the observed variations represents global changes in the carbon distribution between biosphere and ocean.The ¹³C/¹²C ratios of N. pachyderma in the North Atlantic cores display larger regional variations at 18 kyrs. B.P. than at present. To explain these differences, we have plotted the 18 kyrs. B.P. δ¹³C values of N. pachyderma from 17 cores distributed N of 40°N. Comparison with published surface water temperature distribution at 18 kyrs. B.P. indicates that a strong divergent cyclonic cell, centered approximatively 55°N and 15°W, was active during most of the last ice-age maximum This hydrology, analogous to the present Weddell Sea, explains the published evidences of bottom water formation, if located on the northern flank of the gyre, and the strong polar front on the southern flank, probable location of intermediate water formation.     

Native gibberellins and the metabolism of [14C]gibberellin A53 and of [17-13C, 17-3H2]gibberellin A20 in tassels of Zea mays

The native hormones from tassels of maize (Zea mays) were re-investigated. The previous identification by GC/SIM of GA₁, GA₈ and GA₂₉ in normal tassels was confirmed by full GC/MS scans at the correct Kovats retention indices. In tassels of dwarf-1 mutants, GA₄₄,^?GA₁₉, GA₁₇, GA₂₀ and the 16,17-dihydro, 7β,16α,17-trihydroxy derivative of ent-kaurenoic acid were identified by GC/MS. Gibberellin A₁ was not found in the mutant tassels. [¹⁴C]Gibberellin A₅₃ was fed to tassels of the dwarf-5 mutant. In the ethyl acetate-soluble acidic fraction from the feeds, [¹⁴C]GA₄₄ was identified by GC/MS; [¹⁴C]GA₁₉ and [¹⁴C]GA₂₉ were identified by GC/SIM. The GA₂₉ is probably a metabolite of the feeds because the dwarf-5 mutant is known to control the step copalyl pyrophosphate to ent-kaurene in the maize GA-biosynthetic pathway and because GA₂₉ was not identified in a control experiment. The n-butanol fractions obtained from the feeds were shown, by GC/MS, to contain [¹⁴C]GA₅₃ after hydrolysis, suggesting that conjugated [¹⁴C]GA₅₃ is a major metabolite from GA₅₃ feeds. [17-¹³C, 17-³H₂]Gibberellin A₂₀ was fed to normal, dwarf-1 and dwarf-5 tassels. In each case, analysis of the purified ethyl acetate-soluble acidic extracts by GC/MS led to the identification of [¹³C]GA₂₉ and unmetabolized [¹³C]GA₂₀ in which no ¹³C-isotope dilution was observed.     

13C nuclear magnetic resonance observations on the interaction between p-amidinophenylpyruvic acid and trypsin

The formation of an enzyme-inhibitor adduct between bovine trypsin and [2-¹³C]p-amidinophenylpyruvic acid has been investigated by ¹³C NMR spectroscopy. The observation of a resonance at 100.8 ppm demonstrates that the hemiketal formed between the hydroxyl of serine-195 and the 2-¹³C carbon of p-amidinophenylpyruvic acid is sp³ hybridized with no significant deviation from tetrahedral geometry. It is shown that stabilization of the hemiketal oxyanion if it occurs is less effective than in chloromethylketone inhibitor complexes. The tetrahedral adduct is stable from pH 3 to 8. The mechanisms of breakdown of the tetrahedral adduct at pH extremes are discussed.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol

Divinyl-13²,17³-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The ¹H NMR spectra in CDCl₃ showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C13² and C17³ positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.     

Fungal macrolides: Structure determination and biosynthesis of achaetolide,a lactone from Achaetomium cristalliferum

The structure of a new ten-membered lactone, achaetolide, isolated from cultures of Achaetomium cristalliferum is deduced from its mass and NMR spectra and from the study ofsomederivatives. The ¹³C NMR spectra of achaetolide enriched with [1-¹³C], [2-¹³C] and [1, 2-¹³C] acetate established its formation from eight intact acetate units via a precursor octaketide chain.     

13C-NMR study of the glucose synthesis pathways in the bacterium Chlorobium thiosulfatophilum

Photoassimilation of ¹³CO₂ and acetate by the photosynthetic bacterium Chlorobium thiosulfatophilum was investigated using ¹³C-NMR as the method for determination of the labelling pattern of the glucose synthesized by the bacterium. Metabolic pathways functioning in the bacterium were identified by analysis of the multiplet structure of the spectra of tightly coupled systems. The labelling pattern showed that glucose was synthesized in C. thiosulfatophilum mainly by the gluconeogenesis pathway. In agreement with previous investigations, the reserve polysaccharide of C. thiosulfatophilum was shown to be polyglucose, with the glucose units linked predominantly by (1→4)α-glucosidic linkages.     

Isolation of glucosinolates and the identification of o-(α-l-rhamnopyranosyloxy)benzylglucosinolate from Reseda odorata

A method has been developed for the quantitative isolation of glucosinolates by ion-exchange chromatography and high voltage electrophoresis avoiding strongly alkaline and acidic conditions. The compounds were identified by ¹H and ¹³C NMR spectroscopy and through the products arising from enzymatic, acid and alkaline hydrolysis. The method is well suited for the isolation and identification of glucosinolates containing aglucone parts which produce non-volatile compounds on enzymatic hydrolysis. The method has been used in the isolation and identification of 2-hydroxy-2-methylpropylglucosinolate from Reseda alba, 2-hydroxy-2-phenylethylglucosinolate from R. luteola and a new glucosinolate, o-(α-l-rhamnopyranosyloxy)benzylglucosinolate, occurring in R. odorata. The glucosinolate content in different parts of this plant has been determined and the metabolism of glucosinolates is briefly discussed.     

Fractionation of stable isotopes in the Arctic marine copepod Calanus glacialis: Effects on the isotopic composition of marine particulate organic matter

Fractionation of δ¹³C and δ¹⁵N between food, consumer, and faecal pellets was studied in the Arctic marine copepod Calanus glacialis Jaschnov, fed with isotopically distinct algal monocultures. Temporal variations in δ¹³C and δ¹⁵N of copepods that were fed ice algae and phytoplankton followed those of a control group consisting of starved animals. There were no significant trends in the δ¹³C and δ¹⁵N values of copepods that were starved for 42 days, suggesting that the isotopic composition of non-lipid body tissues is unaffected by the metabolic processes during prolonged periods of starvation. The stable isotopic composition of starved copepods therefore seems to reflect food consumed during the previous period of feeding and growth. Faecal pellets produced by feeding copepods were depleted in ¹³C and ¹⁵N by 6.3-11.2‰ and 0.7-9.1‰, respectively, relative to the food ingested. These results indicate that faecal pellet production is an important pathway for the trophic fractionation of δ¹³C, whereas other fractionation pathways, such as excretion of ammonia, may be relatively more important for δ¹⁵N. The strong depletion of ¹³C in faecal pellets compared to the food suggests that grazing by herbivorous copepods on primary production adds to the variability of δ¹³C in marine particulate organic matter.     

N-[1-13C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

Determination of the mode of interaction of Mn2+ and Cu2+ with cis-inositol by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Mn²⁺ and Cu²⁺ to the cyclitol, cis-inositol. Resonance linewidths and the electron nuclear relaxation rates [(T₁^e)^?1 values] were used to establish that a unique binding site exists for these metal-ions on this cyclitol involving only the three axial hydroxyl groups. This work may aid in the development of new organometallic complexes used as paramagnetic relaxation agents in magnetic resonance imaging research.     

The metabolism of anatabine to α,β-dipyridyl in Nicotiana species

α,β-Dipyridyl isolated from Nicotiana tabacum plants which had been fed anatabine-[2′-¹⁴C, ¹³C], and then allowed to dry in air for 20 days was radioactive (82% specific incorporation)An examination of its ¹³C NMR spectra established that it was enriched only at C-2, indicative of its direct formation from anatabineThe labelled anatabine was also fed to Nglauca and Nglutinosa plants, which were extracted immediately after harvestingIn these experiments no radioactive α,β-dipyridyl was detected, suggesting that α,β-dipyridyl is an artifact produced by the oxidation of anatabine in the drying leaves of tobaccoAnabasine isolated from the Nicotiana species which had been fed anatabine-[2′- ¹⁴C, ¹³C] was unlabelled, indicating that none of this alkaloid is formed by the reduction of anatabine.     

Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly ¹³C/¹⁵N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive ¹³C/¹⁵N-labeled amino acids. The most cost-effective production of ¹³C/¹⁵N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% ¹³C-glycerol and 0.5% ¹⁵N-ammonium sulfate, supplemented with only 0.025% of ¹³C/¹⁵N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.     

13C NMR spectroscopy of Pyrrolizidine alkaloids

The ¹³C NMR spectra of nine pyrrolizidine alkaloids of the macrocyclic diester type, seven of the corresponding N-oxides and of the parent base retronecine have been recorded and the signals assigned. The 13C NMR signals were found to be sensitive to structural variation in both the diester moiety and the heterocyclic ring system, providing useful information for structural elucidation, particularly when the ¹H NMR spectra may be difficult to interpret.     

The effect of acidification and the combined effects of acidification/lipid extraction on carbon stable isotope ratios for sub-arctic and arctic marine zooplankton species

Stable isotope ratios of carbon (δ ¹³C) in zooplankton are widely applied in ecosystem-level studies of energy flux and trophic interactions. Carbonate (CaCO₃) and lipid content are highly variable both among and within zooplankton species. Such variability can arise from the δ ¹³C-depleted nature of lipids as well as differences in carbon incorporation among tissues (e.g., relative amount of carbonate). Critically, the impact of the common lipid- and carbonate-normalization steps of extraction and acidification is poorly understood and applied in an inconsistent manner. Here, we investigated the effect of lipid extraction and sample acidification (CaCO₃ removal) on δ ¹³C in sub-arctic and arctic marine zooplankton species. Our results indicate that, with the exception of the shelled mollusc Limacina helicina, acidification of samples can be omitted for all other marine zooplankton considered in this study. In the case of L. helicina, δ ¹³C can be corrected for carbonate content using the linear equation developed in this study. In contrast, the δ ¹³C for all species was significantly enriched by a combination of lipid extraction and acidification (up to +4.9 ‰) prior to stable isotope analysis. Our data were used to develop simple, predictive species-specific correction models for lipid-acid-normalized δ ¹³C using C:N and/or untreated δ ¹³C values. Our results indicate that the δ ¹³C value for all species, including those with lower C:N ratios (~3–4), should be corrected for lipid content. We recommend lipid extraction whenever possible, or else the use of species/taxon-specific δ ¹³C lipid-normalization models for accurate determination of carbon sources and dynamics for arctic and sub-arctic marine zooplankton.     

Secondary structure and backbone dynamics of Escherichia coli diacylglycerol kinase,as revealed by site-directed solid-state 13C NMR

To gain insight into secondary structure and backbone dynamics, we have recorded ¹³C NMR spectra of [3-¹³C]Ala-, [1-¹³C]Val-labeled Escherichia coli diacylglycerol kinase (DGK), using cross-polarization-magic angle spinning (CP-MAS) and single-pulse excitation with dipolar decoupled-magic angle spinning (DD-MAS) methods. DGK was either solubilized in n-decyl-β-maltoside (DM) micelle or integrated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Surprisingly, the ¹³C NMR spectra were broadened to yield rather featureless peaks at physiological temperatures, both in DM solution or lipid bilayers at liquid crystalline phase, due to interference of motional frequencies of DGK with frequencies of magic angle spinning (MAS) or proton decoupling (10⁴ or 10⁵ Hz, respectively). In gel phase lipids, however, up to six distinct ¹³C NMR peaks were well-resolved due to lowered fluctuation frequencies (<10⁵ Hz) for the transmembrane region, the amphipathic α-helices and loops. While DGK can be tightly packed in gel phase lipids, DGK is less tightly packed at physiological temperatures, where it becomes more mobile. The fact that the enzymatic activity is low under conditions where motion is restricted and high when conformational fluctuations can occur suggests that acquisition of low frequency backbone motions, on the microsecond to millisecond time scale, may facilitate the efficient enzymatic activity of DGK.