The biosynthesis of trichothecin from acetate- [1,2- 13C2]

The coupling pattern of trichothecin biosynthesized from acetate-[1,2-¹³C₂] is in accord with previous enrichment studies. Multiple labelling was observed. Exogenous acetate has been shown to inhibit the utilization of glucose and the incorporation of radioactivity from pyruvate-[2-¹⁴C] and citrate-[1,5-¹⁴C] into the metabolites. Two pairs of ¹³C NMR assignments are interchanged.     

Biosynthesis of the sesquiterpenoid,capsidiol, in sweet pepper fruits inoculated with fungal spores

Capsicum frutescens fruits inoculated with spore suspensions of Monilinia fructicola incorporated 1–4% of sodium acetate-[2-¹⁴C] or RS-mevalonolactone-[2-¹⁴C] into the phytoalexin, capsidiol. Labelled capsidiol was characterized by GC-RC, TLC-RC, gel chromatography (in conjunction with liquid scintillation counting) and GC-MS. The mode of incorporation of sodium acetate-[1,2-¹³C₂] into capsidiol, as indicated by the pattern of ¹³C-¹³C coupling from ¹³C NMR data, supports the hypothesis that the angular methyl group of the capsidiol skeleton arises by migration from the C-10 position of a eudesmane-type intermediate.     

In vivo Biosynthesis of isopentenylacetophenones in Eupatorium rugosum

The biosynthesis of dehydrotremetone in Eupatorium rugosum has been investigated by feeding radioactive precursors to intact plants. The carbon atoms of acetate-[1-¹⁴C] and acetate-[2-¹⁴C] were identified in dehydrotremetone by degradation of the molecule. From the pattern of labeling it was concluded that the acetophenone moiety was derived from acetate via the polyacetate pathway. From the incorporation of mevalonate it appeared that the furan ring and its side chain were formed from an isoprenoid compound. Potential aromatic intermediates were chemically synthesized and also fed to plants but only tremetone was found to be efficiently incorporated into dehydrotremetone. Neither 4-hydroxyacetophenone nor 4-hydroxy-3[isopenten-(2)-yl]-acetophenone were efficiently incorporated into dehydrotremetone.     

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′.     

Biosynthesis of monoterpenes in plants from 14C-labelled acetate and CO2

Degradation of (+)-isothujone biosynthesized by Tanacetum vulgare or Thuja plicata from acetate-[1-¹⁴C], -[2-¹⁴C] and -[2-³H₃] or from CO₂-[14C] at physiological concentration revealed a pattern of asymmetric labelling whereby tracer predominantly (72–98% resided in that part of the skeleton derived from IPP. This is similar to the patterns previously obtained for uptake of MVA-[2-¹⁴C] but differed from those reported in other species with acetate-[¹⁴C] as precursor. Within the IPP-derived moiety the 3 parts derived from acetate units were not equivalently labelled. Partial degradations of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium after uptake of ¹⁴C-labelled acetate or CO₂ showed that the C-2 units of the skeletons of these monoterpenes were also labelled to widely differing extents and these patterns persisted over a range of feeding and seasonal conditions. These results suggest that metabolic pools of acetyl-CoA and/or acetoacetyl-CoA exist in these plants. The general occurrence of such pools and the consequent nonequivalent labelling patterns in secondary metabolism could invalidate biosynthetic conclusions drawn from partial degradations of labelled natural products.     

Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are discussed.     

C nuclear magnetic resonance study of acetate incorporation into malate during ca-uptake by isolated leaf tissues

¹³C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from ¹³C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-¹³C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-¹³C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a ¹³C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin.     

Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC₅₀ concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [¹⁴C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K⁺-efflux and greater leakage of intracellular [³²P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by C turnover from hyperpolarized [1-C]acetate to [1-C]acetylcarnitine

Background

Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle.

Methods

Hyperpolarized [1-¹³C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The ¹³C magnetic resonance signals of [1-¹³C]acetate and [1-¹³C]acetylcarnitine were recorded in vivo for 1 min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3 s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios.

Results

Although separated by two biochemical transformations, a kinetic analysis of the ¹³C label flow from [1-¹³C]acetate to [1-¹³C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were K_M = 0.35 ± 0.13 mM and V_max = 0.199 ± 0.031 μmol/g/min.

Conclusions

The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results.

General significance

This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.     

Regularity of isoprenoid biosynthesis in the ether lipids of archaebacteria

The location of paired and unpaired ¹³C atoms in the 16,16′-biphytanyl components of the lipids of Caldariella acidiophila following incorporation of acetate-[1,2-¹³C₂] shows that the overall process of isoprenoid biosynthesis in this archaebacterial species follows a normal pattern and that the head-to-head linkage of the two tetraprenyl chains occurs stereoselectively.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

Biosynthesis of Fatty acids by a soluble extract from developing soybean cotyledons

Fractionation of developing soybean cotyledons into cellular components demonstrates that most of the activity necessary to incorporate acetate-1-¹⁴C into lipid remains in the supernatant from a 198,000g spin for 1 hr. The system studied is dependent upon ATP, CoA, and CO₂. Concentrations of ATP greater than 4 × 10⁻³m are inhibitory, while 1 × 10⁻⁴m CoA is needed for optimal activity. Avidin inhibition of acetate incorporation into lipid could be reversed by biotin. Studies indicated that NADPH is a better source of reducing power than NADH. The system studied is inhibited by p-chloromercuribenzoic acid and this inhibition can be reversed by an excess of GSH. The system studied shows maximum activity in tris buffer at pH 8.6 or in glycine buffer, pH 9.4.     

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.     

The fate of C-20 in C19 gibberellin biosynthesis

Experiments with ent-kaur-16-ene-[¹⁴C], prepared biosynthetically from sodium acetate-[2-¹⁴C], have shown that the C-20 carbon atom of the C₂₀ gibberellins is evolved as carbon dioxide during the biosynthesis of the C₁₉ gibberellins by Gibberella fujikuroi.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

Long Chain (C(20) and C(22)) Fatty Acid Biosynthesis in Developing Seeds of Tropaeolum majus: AN IN VIVO STUDY

The storage triacylglycerols of nasturtium (Tropaeolum majus) seeds are composed principally of cis-11-eicosenoate and cis-13-docosenoate. To investigate the biosynthesis of these C₂₀ and C₂₂ fatty acids, developing seed tissue was incubated with various ¹⁴C-labeled precursors. Incubation with [1-¹⁴C]acetate produced primarily cis-11-[1-¹⁴C]eicosenoate and cis-13-[1,3-¹⁴C]docosenoate in the triacylglycerol fraction, the odd-carbon [U-¹⁴C]oleate also formed from [¹⁴C] acetate was in the polar lipid fraction. Kinetic data showed that this oleate was not channeled into cis-11-eicosenoate nor cis-13-docosenoate over a 24-hour period. Under suitable conditions, nasturtium seed could also produce [¹⁴C]stearate, [¹⁴C]eicosenoate, and [¹⁴C]docosenoate from [1-¹⁴C]acetate. The results are discussed in terms of the number of pathways producing fatty acids. From pool size and other considerations, the results can be rationalized only in terms of different de novo systems for oleate biosythesis, one supplying oleate for incorporation into phospholipids and the other supplying oleate for chain elongation and subsequent esterification into triacylglycerols. Because of the probable heterogeneous nature of the seed tissue, it is not known if these two systems are operating in different cell types, in the same cell type at different stages of development, or in the same cell type concurrently.     

Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo

Rat liver microsomal incubation systems containing the free radical spin trap, phenyl-t-butyl nitrone, as well as an NADPH generating system and [¹³C]CCl₄ (90 atom % ¹³C) produce electron spin resonance spectra consistent with that expected for a trichloromethyl-phenyl-t-butyl nitrone adduct. This same spectrum is observed in a lipid extract of the liver from a rat orally administered [¹³C]CCl₄ as well as in a solution of phenyl-t-butyl nitrone and [¹³C]CCl₄ irradiated with ultraviolet light.     

In vivo study of cholesterol turnover in tissues of adult sows

The in vivo study of free and esterified cholesterol turnover was carried out in 15 tissues of adult Large White sows maintained at a constant weight for 10–12 weeks. They received a single intravenous injection either of [1-¹⁴C] acetate, or of an autologous red cell suspension or of plasma, previously labelled in vitro (for red cells) or in vivo (for plasma) with tritiated cholesterol.The tissues can be separated into four groups according to their relative rate of free cholesterol exchange between plasma and tissues. The liver and the lungs have a very fast exchange rate whereas the brain and the spinal cord have a very slow one. The whole lipoprotein particle transfer — an exclusive model for the esterified cholesterol transport from plasma to tissues — has been found in all sow tissues. When [1-¹⁴C] acetate is used as a substrate for cholesterol synthesis, lungs, adrenal glands and heart do not seem — or at an extremely low rate — to convert acetate into cholesterol whereas an intense cholesterol synthesis takes place in the small intestine. Its contribution to cholesterol synthesis in sows — taking into account the cholesterol transfer processes — reaches 70 per cent.     

A new biosynthetic pathway of porphyrins from isopropanol

A new biosynthetic pathway, which can produce both vitamin B₁₂ and large amounts of porphyrins from isopropanol, was identified in Arthrobacter hyalinus using carbon-13 stable isotope tracer techniques and carbon-13 nuclear magnetic resonance (¹³C-NMR) spectroscopy. Studies on the incorporation of [2-¹³C]isopropanol, [1- or 2-¹³C]sodium acetate, l-[1-¹³C]glutamate, and [1-, 2-, 3-, 4-, 5-¹³C]5-aminolevulinic acid into uroporphyrinogen III showed that isopropanol was metabolized into uroporphyrinogen III through acetyl CoA and that 5-aminolevulinic acid was produced from l-glutamic acid and not via Shemin's pathway.