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1.
Etiolated maize and soybean seedlings were grown for several days in atmospheres enriched with O18. Hydroxyproline subsequently isolated from the seedlings by column and thin-layer chromatography was labeled with excess O18, but proline was not. Control experiments in which seedlings were grown in H2O18 and unlabeled atmospheres demonstrated that neither proline nor hydroxyproline was labeled with excess O18. It was concluded that oxygen fixation is an essential feature of hydroxyproline biosynthesis in these seedlings, and that the hydroxyl oxygen atom in hydroxyproline is derived from molecular oxygen and not from water; similar results have been reported previously for sycamore cell suspensions.  相似文献   

2.
The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of 18O2 or in a medium containing H218O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate (t12 = 9.5–336 min) and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia.  相似文献   

3.
The biosynthetic origin of 9-phenylphenalenones and the sequence according to which their oxygen functionalities are introduced were studied using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). 13C-labelled precursors were administered to root cultures of Anigozanthos preissii, which were simultaneously incubated in an atmosphere of 18O2. Two major phenylphenalenones, anigorufone and hydroxyanigorufone, were isolated and analyzed by spectroscopic methods. Incorporation of 13C-labelled precursors from the culture medium and 18O from the atmosphere was detected. O-Methylation with 13C-diazomethane was used to attach 13C-labels to each hydroxyl and thereby dramatically enhance the sensitivity with which NMR spectroscopy can detect 18O by means of isotope-induced shifts of 13C signals. The isotopologue patterns inferred from NMR and HRESIMS analyses indicated that the hydroxyl group at C-2 of 9-phenylphenalenones had been introduced on the stage of a linear diarylheptanoid. The oxygen atoms of the carbonyl and lateral aryl ring originated from the hydroxyl group of the 4-coumaroyl moiety, which was incorporated as a unit.  相似文献   

4.
Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (−)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used 18O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under 18O2, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H218O. Pisatin synthesized by pea pods in the presence of 18O2 or H218O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H2O and not from O2. Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.  相似文献   

5.
The interaction of water with dehydroascorbic acid was examined by incubating dehydroascorbic acid and ascorbic acid in18O-labeled water for various amounts of time and then oxidizing the products with hydrogen peroxide or reducing the products with mercaptoethanol, with analysis by gas chromatography mass spectrometry. Based on mass changes, dehydroascorbic acid readily exchanged three oxygen atoms with H218O. When mercaptoethanol was used to reduce dehydroascorbic acid (which had been incubated in H218O) to ascorbic acid, the newly formed ascorbic acid also contained three labeled oxygen atoms. However, ascorbic acid incubated in H218O for the same amount of time under identical conditions exchanged only two labeled oxygen atoms. Electron impact mass spectrometry of derivatized ascorbic acid created a decarboxylation product which had only two labeled oxygen atoms, regardless if 3-oxygen-labeled or 2-oxygen-labeled ascorbic acid was the parent compound, isolating the extra oxygen addition to carbon 1. These data suggest that dehydroascorbic acid spontaneously hydrolyzes and dehydrates in aqueous solution and that the hydrolytic-hydroxyl oxygen is accepted by carbon 1. Ascorbic acid, on the other hand, does not show this same tendency to hydrolyze.  相似文献   

6.
[4-14C]Cholesterol was incubated with an adrenocortical preparation in the presence of 16O2 and 18O2 devoid of significant 16O18O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of me 289, 291, and 293 (comprising the C8 C-20 to C-27 side-chain and containing, respectively, 16O2, 16O18O, and 18O2) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of me 291 would have been absent.  相似文献   

7.
Heavy water (H218O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the 18O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% 18O for DNA extracted from soil incubated with the addition of H218O. Four experiments were conducted to test how the atom% 18O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% 18O of added H2O. In the time series experiment, the atom% 18O of DNA increased linearly (R 2 = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R 2 = 0.991, p = 0.006) between the log10 of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log10 of the atom% 18O of DNA. For the experiment where soil moisture was manipulated, the atom% 18O of DNA increased with higher soil moisture until soil moisture reached 30%, above which 18O enrichment of DNA declined as soils became more saturated. When the atom% 18O for H2O added was varied, there was a positive linear relationship between the atom% 18O of the added water and the atom% 18O of the DNA. Results indicate that quantification of 18O incorporated into DNA from H218O has potential to be used as a proxy for microbial growth in soil.  相似文献   

8.
Curcumin is a dietary anti-inflammatory and chemopreventive agent consisting of two methoxyphenol rings connected by a conjugated heptadienedione chain. Curcumin is unstable at physiological pH and rapidly degrades in an autoxidation reaction to a major bicyclopentadione product in which the 7-carbon chain has undergone oxygenation and double cyclization. Early degradation products (but not the final bicyclopentadione) mediate topoisomerase poisoning and possibly many other activities of curcumin, but it is not known how many and what autoxidation products are formed, nor their mechanism of formation. Here, using [14C2]curcumin as a tracer, seven novel autoxidation products, including two reaction intermediates, were isolated and identified using one- and two-dimensional NMR and mass spectrometry. The unusual spiroepoxide and vinylether reaction intermediates are precursors to the final bicyclopentadione product. A mechanism for the autoxidation of curcumin is proposed that accounts for the addition and exchange of oxygen that have been determined using 18O2 and H218O. Several of the by-products are formed from an endoperoxide intermediate via reactions that are well precedented in lipid peroxidation. The electrophilic spiroepoxide intermediate formed a stable adduct with N-acetylcysteine, suggesting that oxidative transformation is required for biological effects mediated by covalent adduction to protein thiols. The spontaneous autoxidation distinguishes curcumin among natural polyphenolic compounds of therapeutic interest; the formation of chemically diverse reactive and electrophilic products provides a novel paradigm for understanding the polypharmacological effects of curcumin.  相似文献   

9.
《BBA》1987,893(3):564-571
In the present paper we analyzed the properties of the S3-state in the filamentous cyanobacterium Oscillatoria chalybea by mass spectrometry. In this organism a substantial O2-signal due to a single flash is observed even after extensive dark adaptation (20 min). This signal can be measured by mass spectrometry as well as amperometrically on an oxygen electrode and is not due to an interference of respiratory and photosynthetic electron transport in the prokaryotic membrane. The mass spectrometric analysis shows that, if S3 is generated by two flashes in a medium containing only H216O, addition of H218O and subsequent firing of a third flash yields O2 evolution labelled with 18O. It appears that the isotopic composition of the O2 evolved corresponds to the isotopic composition of the water in the suspension. This experiment shows that water oxidation does not proceed via an oxygen precursor or water derivatives bound to the S3-state. This conclusion has been reached shortly before ours by Radmer and Ollinger [15] in the reverse marker experiment. From our study with O. chalybea it appears that freshly generated S3 can be distinguished from metastable S3 by the mass spectrometric method. It looks as if in contrast to freshly generated S3 metastable S3 contained bound unexchangeable water or an oxidized water derivative.  相似文献   

10.
The cyanobacterium Acaryochloris marina was cultured in the presence of either H218O or 18O2, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H218O, newly synthesized Chl a and d, both incorporated up to four isotopic 18O atoms. Time course H218O labeling experiments showed incorporation of isotopic 18O atoms originating from H218O into Chl a, with over 90% of Chl a 18O-labeled at 48 h. The incorporation of isotopic 18O atoms into Chl d upon incubation in H218O was slower compared with Chl a with ∼50% 18O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of 18O2 gas, one isotopic 18O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H218O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic 18O atoms derived from molecular oxygen (18O2) was observed in the extracted Chl d, and the percentage of double isotopic 18O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C31-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.  相似文献   

11.
Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% 18O2 and 80% N2 indicates that one atom of 18O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing 18O2 indicates that one atom of 18O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress.

  相似文献   

12.
Incubation of epicubenol synthase with farnesyl pyrophosphate in the presence of 11.1 atom% H2(18)O gave epicubenol (2) in which the hydroxyl oxygen atom was shown to be derived exclusively from water, as established by GC-selected ion monitoring MS of the derived TMS-epicubenol derivative (15).  相似文献   

13.
In a previous study where rat skin collagen was labeled with 18O in the hydroxyl group of the collagen hydroxyproline we noticed that the decay rate of this label was much faster than had been observed when the skin collagen hydroxyproline was labeled with 3H in the prolyl ring. In this study a rat was labeled concurrently with [18O2] and [3H] proline and the rate of decline of both labels was determined in rat skin collagen hydroxyproline. After correction for growth dilution of the skin collagen the [18O] hydroxyproline was found to have a half-life of 27 days while the [3H] hydroxyproline had a half-life of 53 days. The decay rate of the [18O] hydroxyproline represents the true turnover rate of collagen since there is no possibility of recycling this label. Hence, the difference between this and the [3H] hydroxyproline decay rate is due to recycling of l-[3H] proline into new collagen. The efficiency of recycling of proline from catabolized collagen into new collagen was about 93%.  相似文献   

14.
Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H218O did not indicate any 18O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R. eutropha JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was 18O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.  相似文献   

15.
Mechanistic studies on C-19 demethylation in oestrogen biosynthesis   总被引:9,自引:1,他引:8       下载免费PDF全文
Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O2 and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with 2H in addition to 18O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[18O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent 16O substrates were aromatized under 18O2, the formic acid from both substrates contained one atom of 18O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O2 is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10β-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10–C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.  相似文献   

16.
Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium   总被引:11,自引:9,他引:2       下载免费PDF全文
Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in 18O2. It was found that in stressed leaves three atoms of 18O from 18O2 are incorporated into the ABA molecule, and that the amount of 18O incorporated increases with time. One 18O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in 18O2 shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more 18O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, 18O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied 14C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional 18O incorporated during 8′-hydroxylation of ABA to phaseic acid.  相似文献   

17.
Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H218O showed incorporation of up to three 18O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H2O. Toxin formed in the presence of 18O molecular oxygen incorporated up to six 18O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H218O and 18O2 labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.  相似文献   

18.
Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of 16O2, 18O2, H216O, and H218O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of 18O-compounds. Incorporation of one 18O atom in ODTD was found if the cleavage reaction was performed in the presence of 18O2 and H216O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H218O in the presence of 16O2 indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of 18O2, H216O, and alcohol dehydrogenase/NADH, incorporation of two atoms of 18O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.  相似文献   

19.
Rats were exposed to air containing 18O2 at atmospheric pressure. In vivo incorporation of 18O in brain homovanillic acid (HVA) was determined by gas chromatography-mass spectrometry. One 18O atom was incorporated into each molecule of HVA indicating that tyrosine is the predominant precursor of brain dopamine and that the oxygen in the 3-position is of atmospheric origin. Intraperitoneal administration of 18O-enriched water did not alter the 18O content of brain HVA Mass fragmentography (2) was used to measure the increase in 18O and the decrease in 16O in HVA from rat brain over several hours of exposure to an 18O enriched atmosphere. These experiments demonstrate the possibility to pulse label brain dopamine and its metabolites by in vivo inhalation of stable oxygen isotopes. The procedure should be useful for quantitative determinations of the turnover of brain dopamine in animals and man.  相似文献   

20.

Introduction

Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.

Objectives

Pathway-specific labeling using isotopic molecular oxygen (18O2) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18O2-metabolome analysis.

Methods

18O2 labeled whole Medicago truncatula seedlings were prepared using 18O2-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).

Results

A total of 511 peaks labeled by 18O2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by 18O2. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSn analysis, we estimated that 18O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.

Conclusion

Our finding indicated that 18O2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.
  相似文献   

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