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1.
Meadowfoam (Limnanthes spp.) species are unique in that their seeds are rich in the unusual fatty acids Δ5-eicosenoic acid (C20:1Δ5) and the diene, C22:2Δ5, Δ13. Previously the cloning of Δ5 desaturase (Des5) and fatty acid elongase 1 (FAE1) meadowfoam genes and their expression in soybean were reported. Here, we present the first successful expression of the Limnanthes Des5 in yeast, resulting in the desaturation of C16:0, C18:0 and C20:0 to their corresponding cis Δ5 isomers. In soybean (Glycine max L.), Limnanthes Des5/FAE1 double transformant somatic embryos fed with radiolabeled C14:0 or C16:0 could elongate these substrates to C18:0, C20:0 and C22:0 and C24:0. However, radiolabeled C18:1Δ9 or C20:1Δ11 were not elongated to their respective monounsaturated very long-chain products, confirming that the cloned Limnanthes FAE1 homolog gene product was specific for elongating saturated fatty acids. To understand better the biosynthetic pathway for C22:2Δ5, Δ13, soybean somatic embryos transformed with the Des5 cDNA were fed in culture with 〚1-14C〛C 22:1Δ13 fatty acid, which resulted in the biosynthesis of 〚1-14C〛-labeled C22:2Δ5, Δ13. Cell-free preparations enriched with detergent-solubilized Δ5 desaturase activity extracted from both developing meadowfoam seeds and from Des5 transgenic soybean embryos, produced 14C-22:2Δ5, Δ13 when supplied with 〚1-14C〛 C22:1-CoA. Thus, both the in vivo and in vitro experiments showed that the biosynthesis of C22:2Δ5, Δ13 can occur in somatic soybean embryos transformed with the Limnanthes Des5 cDNA, and confirmed that the pathway for C22:2 biosynthesis in meadowfoam involves further desaturation of erucoyl-CoA by a Δ5-regiospecific desaturase.  相似文献   

2.
The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C20 and C22 fatty acids, which contain an unusual Δ5 double bond. When [1-14C]acetate was incubated with developing seed slices, 14C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [14C]acetate and [14C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[14C]11-eicosenoate, cis-13-[14C]docosenoate, and cis-5,cis-13-[14C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C20 and C22 acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-14C-labeled free fatty acids. Using [1-14C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.  相似文献   

3.
[17-13C,3H]-Labeled gibberellin A20 (GA20), GA5, and GA1 were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). 13C-Labeled GA29, GA8, GA5, GA1, and 3-epi-GA1, as well as unmetabolized [13C]GA20, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-13C, 3H]GA20 to all three genotypes. 13C-Labeled GA8 and 3-epi-G1, as well as unmetabolized [13C]GA1, were identified by GC-SIM from feeds of [17-13C, 3H]GA1 to all three genotypes. From feeds of [17-13C, 3H]GA5, 13C-labeled GA3 and the GA3-isolactone, as well as unmetabolized [13C]GA5, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-13C, 3H]GA5 to [13C]GA1, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA20, as follows: (a) GA20 → GA1 → GA8; (b) GA20 → GA5 → GA3; and (c) GA20 → GA29. The in vivo biogenesis of GA3 from GA5, as well as the origin of GA5 from GA20, are conclusively established for the first time in a higher plant (maize shoots).  相似文献   

4.
《Carbohydrate research》1986,147(2):247-264
l-(1-13C, 5-2H)Arabinose (6D) and l-(2-13C, 5-2H)arabinose (8D) have been synthesized by degradation of 2,3-O-isopropylidene-α-l-rhamnofuranose (2) to l-(4-2H)erythrose (,D), with subsequent chain elongation to 6D plus l-(1-13C, 5-2H)ribose (7D), the latter being converted into 8D. Intermediates were identified by complete assignment of the 13C chemical shifts employing carbon-carbon and carbon-deuterium coupling constants, deuteration shifts, differential isotope-shifts, and deuterium spectra. The anomeric carbon atoms of 2 and 2,3-O-isopropylidene-l-(1-2H) erythrose (4D) gave only single 13C resonances, suggesting that these two compounds exists in only one major anomeric configuration, clarifying previously reported work. The synthesis of 2,3-O-isopropylidene-l-(1-2H)rhmanitol (3D) facilitated the assignment of the signals in the 13C spectra of the nondeuterated analog. Specific deuterium-enrichment and the observed carbon-deuterium coupling (1JC,D ∼22 Hz) not only served to identify the deuterated carbon atom unambiguously in 3 but also permitted assignment of closely spaced resonances. The deuterium spectrum of 2,3-O-isopropylidene-l-(1-2H)erythrofuranose (4D) showed only a single resonance, indicating preponderance of one anomer, in accord with the observation of a single C-1 resonance in the 13C spectrum.  相似文献   

5.
In addition to plantarenaloside and stansioside, a new iridoid glucoside with a formyl group at C-4 has been isolated from Tecoma stans. The new glucoside was shown to be 5-deoxystansioside by 13C NMR and 1H NMR spectroscopy.  相似文献   

6.
The seasonal changes of pigments and stable carbon isotope composition (δ13C values) were investigated in needles of Qilian juniper (Sabina przewalskii Kom.) from two typical sites, one drier and the other wetter, in the Qilian Mountains, China. The anthocyanins and rhodoxanthin content from both sites were much higher in winter than in summer. Plant needles contained more carotenoids and xanthophylls in winter at the wetter site, while no seasonal difference appeared at the drier site. However, lower chlorophyll content and higher proline and δ13C were observed at the drier site. Average tree height was shorter at the drier site trees than at the wetter site. Our results suggested that under natural conditions, pigmentation in S. przewalskii may serve to improve the energy balance of the photosynthetic apparatus under cold and drought stresses. Proline and δ13C could be used as drought indicators for S. przewalskii.  相似文献   

7.
This paper presents a 13C CP/MAS NMR study of the melanin pigments obtained through natural synthetic origins: sepia-melanin from squid ink and three synthetic 5,6-dihydroxyindole-melanins prepared using different non-enzymatic oxidation pathways. The synthetic pigments can be distinguished from natural melanin by the absence of aliphatic carbons, thereby confirming the unreacted 3,4-dihydroxyphenylalanine and the proteinaceous origins of the aliphatic resonances in natural eumelanin. The spectra of selected non-protonated carbon resonances and those with only protonated carbon signals led to a quantitative analysis. An auto-oxidative experiment using a synthetic melanin, over a period of 130 h, has shown an usually slow disappearance of hydrogen peroxide formed in situ. The 13C-NMR spectrum of the insoluble oxidized synthetic melanin compared to that before auto-oxidation clearly demonstrates that the oxidation process is associated with chemical changes within the pigment; i.e., carbonyl functional group formation and an increase of the non-protonated carbons fraction.  相似文献   

8.
1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4–8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with 14C by using 5-amino[4-14C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major 14C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with 59Fe by using 59FeCl3, norethindrone administration resulted in the detection of 59Fe-labelled green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H2SO4. These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.  相似文献   

9.
Developing chloroplasts isolated from cucumber (Cucumis sativus L. var Beit Alpha) cotyledons are capable of incorporating [14C]5-aminolevulinic acid into chlorophyll (Chl) b and Chl a when incubated under photosynthetic illumination. Thin layer chromatography and high pressure liquid chromatography were employed to analyze the pigments. The specific radioactivity in Chl a was over three times higher than that found in Chl b. Both Chl a and b synthesizing activities in organello decayed rapidly at approximately the same rate. We conclude that concomitant synthesis of Chl a/b-binding apoprotein is not required for Chl b synthesis.  相似文献   

10.
Pseudomonas fluorescens GcM5-1A, isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, was cultured in Luria Broth medium (LB). The clarified culture was extracted with ethyl acetate, and two dipeptides were purified from the extract. The chemical structures of 1 and 2 were identified as cyclo(-Pro-Val-)and cyclo(-Pro-Tyr-), respectively, by MS, 1H NMR, 13C NMR,1H-1H COSY, 1H -13C COSY spectra. Bioassay results showed that the two compounds were toxic to both suspension cells and seedlings of Pinus thunbergii, which may offer some clues to research the mechanism of pine wilt disease caused by PWN.  相似文献   

11.
Partially mycoheterotrophic (mixotrophic) plants gain carbon from both photosynthesis and their mycorrhizal fungi. This is considered an ancestral state in the evolution of full mycoheterotrophy, but little is known about this nutrition, and especially about the physiological balance between photosynthesis and fungal C gain. To investigate possible compensation between photosynthesis and mycoheterotrophy in the Mediterranean mixotrophic orchid Limodorum abortivum, fungal colonization was experimentally reduced in situ by fungicide treatment. We measured photosynthetic pigments of leaves, stems, and ovaries, as well as the stable C isotope compositions (a proxy for photosynthetic C gain) of seeds and the sizes of ovaries and seeds. We demonstrate that (1) in natural conditions, photosynthetic pigments are most concentrated in ovaries; (2) pigments and photosynthetic C increase in ovaries when fungal C supply is impaired, buffering C limitations and allowing the same development of ovaries and seeds as in natural conditions; and (3) responses to light of pigment and 13C contents in ovaries shift from null responses in natural conditions to responses typical of autotrophic plants in treated L. abortivum, demonstrating photoadaptation and enhanced use of light in the latter. L. abortivum thus preferentially feeds on fungi in natural conditions, but employs compensatory photosynthesis to buffer fungal C limitations and allow seed development.  相似文献   

12.
AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.  相似文献   

13.
Abstract

The protected (4a-9a) and deprotected (4b-6b, 8b) glucuronides of 5-fluorocytosine and 5-fluorouracil were synthesized and characterized by mass spectrometry and 19F, 1H and 13C NMR. The substitution position of the sugar moiety on the pyrimidine ring was determined from the 13C NMR chemical shift of the C1′ of the sugar. The α or β configuration of the glucuronide linkage was assigned on the basis of the value of the coupling constant between H1′ and H2′ of the sugar.  相似文献   

14.
A synthesis of 5-thio-D-galactose, in the form of its crystalline, anomeric methyl glycopyranosides, is described. Compounds prepared as intermediates included ethyl 2,3-di-O-(tert-butyldimethylsilyl)-5,6-O-carbonyl-β-D-galactofuranoside, the corresponding 5,6-dideoxy-5,6-epithio derivative, and ethyl 2,3,6-tri-O-acetyl-5-S-acetyl-5-thio-β-D-galactofuranoside. On methanolysis, the latter afforded methyl 5-thio-α-D-galactopyranoside which, in turn, was transformed into methyl 5-thio-β-D-galactopyranoside. Acetolysis proved to be less satisfactory for incorporation of the sulfur atom into a pyranose ring-form. Characteristics of the 13C-n.m.r. spectra of derivatives of 5-thio-D-galactose are described, including the fact that 1JC,H values for the anomeric pyranosides differ by only 1–3 Hz, as compared with ≈ 10 Hz for their oxygen analogs.  相似文献   

15.
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.  相似文献   

16.
Proline metabolism is implicated in plant responses to abiotic stresses, including the chilling stress. During proline catabolism, the two-step oxidation of proline is performed by the continuous actions of proline dehydrogenase (ProDH), which produces Δ1-pyrroline-5-carboxylate (P5C), and P5C dehydrogenase (P5CDH), which oxidizes P5C to glutamate. The Arabidopsis thaliana chilling mutants chs1 and chs2 are sensitive to chilling temperatures of 13–18°C. For a better understanding of Arabidopsis responses to chilling stress, 4-week-old wild-type (WT) and chs1 and chs2 lines, with three plants in each group, were subjected to chilling stress (13°C), cold stress (4°C), or remained under normal conditions (23°C); and several factors including the expression of ProDH2 and P5CDH genes, POX (peroxidase) and SOD (superoxide dismutase) activities, as well as MDA and proline contents were examined. Our results showed an increase in the proline content in all lines under chilling conditions. In addition, a greater expression of ProDH2 and a lower expression of P5CDH were observed, leading us to speculate a greater breakdown of proline into P5C and a consequent overproduction of ROS in the ETC cycle. The higher POX and SOD activities and a higher MDA content in chs mutants at 13°C are in line with this speculation. Finally, cold-treated plants (4°C) only showed an increase in proline levels.  相似文献   

17.
The elimination of halide ion from either 5-bromo- or 5-iodo-5,6-dihydrouracil to yield uracil is a slow reaction which, in the case of 5-iodo-5,6-dihydrouracil, is 400 times slower than the enzymatic release of 125I? from 5-[125I]iodouracil. The elimination of HBr from 5-bromo-5,6-dihydrouracil is subject to general base catalysis by tris(hydroxymethyl)aminomethane (k2Tris base = 11 × 10?4M?1 min?1, 37°C, ionic strength 1.0 M). At pH values near and above physiological, both the bromo- and iododihydropyrimidines are subject to hydrolysis of the dihydropyrimidine ring, a reaction which parallels halide elimination to yield uracil. The resulting 2-halo-3-ureidopropionate then cyclizes via intramolecular attack of the ureido oxygen atom to yield halide ion and 2-amino-2-oxazoline-5-carboxylic acid as final products. In dilute hydroxide ion, the kinetics of 5-bromo-5,6-dihydrouracil hydrolysis (25°C, ionic strength 1.0 M) show a change in rate-determining step as a function of increasing hydroxide ion concentration, a result which, as in the case of 5,6-dihydrouracil, can be explained in terms of the formation of a tetrahedral addition intermediate. The data are discussed relative to enzymatically catalyzed halopyrimidine dehalogenation.  相似文献   

18.
The in vitro cultured liverwort Jungermannia subulata produces the unique molecule subulatin. In this study, we examined the incorporation of [1-13C] and [1,2-13C2] glucose, [2-13C] arabinose, [2-13C] caffeic acid, and [1-13C] phenylalanine into subulatin. The trilobatinoic acid C unit of subulatin incorporated 13C atoms from [1-13C] and [1,2-13C2] glucose and from [2-13C] arabinose but not from any other of the other precursors. Based on these results and labeling patterns, the trilobatinoic acid C unit of subulatin appears to be biosynthesized from arabinose-5-phosphate and phosphoenolpyruvate.  相似文献   

19.
32P-labeled oligonucleotides from a pancreatic DNase digest of non-glucosylated T2 gt? DNA have been analyzed by high voltage electrophoresis (both before and after dephosphorylation of the 5′ terminus). T2 gt? oligonucleotides, which contain 5-hydroxymethyl cytosine (hm5C) in place of cytosine (C), have altered electrophoretic mobilities compared to fd DNA oligonucleotides (which contain C). In addition, we have observed that pancreatic DNase exhibits a marked cleavage specificity; i.e., hm5C is the predominant 5′ terminal residue in the hm5C-containing oligonucleotides we have characterized.  相似文献   

20.
A study of the reactions of Mo(CO)5(P(OCH2- CMe2CH20)X) (X = C1, Br) with a variety of nucleophiles of the type HER (E = NH, O, S; R = H, alkyl, aryl) is reported. The mechanism of these reactions is shown to be SN2, and the significantly slower rates of reactions of n-propylamine with the above complexes relative to the rate of reaction of n-propylamine with Mo(CO)5(Ph2PC1) is discussed.The 13C, 17O, 31P and 95Mo NMR data and infrared data for these complexes are presented. Good correlations between chemical shifts of the cis and trans CO13C and trans CO170 NMR resonances, CO infrared stretching force constants and the magnitudes of 1JMop and 2JPC (trans CO) are observed and the reasons for these correlations are discussed.The correlations between the chemical shifts of NMR-active nuclei in the Mo(CO)5(P(OCH2CMe2- CH2O)ER) complexes with the chemical shifts of similar nuclei in the Mo(CO)5(Ph2PER) complexes fange from excellent to very poor. This indicates that the effects of-the P-substituents on the chemical shifts of the NMR-active nuclei in these complexes are not additive.  相似文献   

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