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1.
Biosynthetic studies of the guaianolide-type sesquiterpene lactones 11βH,13-dihydrolactucin-8-O-acetate and 8-desoxylactucin were performed in Agrobacterium rhizogenes—transformed hairy root cultures of blue-flowered lettuce, Lactuca floridana. The 13C NMR spectra of the two guaianolides labelled by incorporation of [1-13C], [2-13C], [1,2-13C2]acetate and [2-13C]mevalolactone showed patterns of enrichment consistent with a previously proposed biogenetic pathway for guaianolide-type sesquiterpene lactones via the acetate-mevalonate-germacradiene route.  相似文献   

2.
The oxidation of [1-14C]linoleate in isolated microsomes from pea leaves was found to be stimulated by NADPH addition. The formation of one of the main metabolites, 12-hydroxy-9(Z)-dodecenoic acid is particularly NADPH-dependent. The predominant products in the absence of NADPH were hydroperoxides and in the presence of NADPH, 12-hydroxy-9(Z)-dodecenoic acid. Exogenous [1-14C]-13-hydroperoxy-9(Z), 11(E)-octadecadieoic acid and [1-14C]-12-oxo-9(Z)-dodecenoic acidwere the efficient precursors of 12-hydroxy9(Z)-dodecenoic acid. It was concluded that 12-hydroxy-9(Z)-dodecenoic acid is formed by NADPH-dependent enzymatic reduction of 12oxo-9(Z)-dodecenoic acid. The observed inhibition of linoleate oxidation in isolated microsomes by CO and metryapone suggests the involvement of cytochrome P-450 in the reaction. The relative contribution of lipoxygenase and monooxygenase activity to linoleate oxidation in microsomes is discussed.  相似文献   

3.
[2S-2-2H]- and [2R-2-2H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-2H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-2H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.  相似文献   

4.
5.
A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1-3H]-Cholesterol and [4-14C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1-3H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1-3H]-cholesterol sulphate, (22R)-22-hydroxy-[22-3H]-cholesterol, and 25-hydroxy-[26-14C]-cholesterol are not incorporated to a significant extent.  相似文献   

6.
Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1-13C)glucose, D-(2-13C)glucose, D-(4-13C)glucose, D-(6-13C)glucose or a mixture of D-(1-13C)glucose and D-(2-13C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. 13C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of 13C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6-13C)glucose was low, compared to that of cellulose and curdlan.  相似文献   

7.
A gas chromatographic–combustion isotope ratio mass spectrometric (GC–C-IRMS) method for the determination of [1-13C]valine enrichments in protein hydrolysates is described. Using a quick derivatization method, δ13C values of the N-methoxycarbonyl methyl ester of valine can be determined from baseline separated GC peaks. Evaluation studies with respect to precision, accuracy, linearity, reduction capacity of the CuO combustion furnace and isotope dilution as a result of derivatization, showed that our GC–C-IRMS system allows robust measurement of enrichments of [1-13C]valine in the range 0 to 1.5 MPE (S.D.±0.01 MPE, n=3). Therefore this method is suited to determine fractional synthetic rates (FSRs) of proteins as low as one-tenth of the FSR of human albumin, in studies using a primed, continuous (6 h) infusion with [1-13C]valine plasma enrichments of approximately 15 MPE and an hourly sampling schedule.  相似文献   

8.
[10D-3H; 3-14C]- and [10L-3H; 3-14C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B4 and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the 3H/14C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A4 whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.  相似文献   

9.
Minoru Otokawa 《BBA》1973,292(3):834-836
An inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis was shown. Pi inhibited the terminal phosphate liberation from [γ-32P]ATP by 30-S dynein and inhibited the conversion of [8-14C]ATP to ADP and AMP by digitonin-extracted cilia.  相似文献   

10.
Nymphs of Rhipicephalus appendiculatus and Hyalomma anatolicum anatolicum were fed on rabbits and maintained in the laboratory to allow moulting and further development. At specified time intervals from 0 to 500 days after engorgement, samples of ticks were ground individually in distilled water within the wells of an agglutination plate. A 0.1 ml aliquot was removed from each and levels of haemin and protein assessed from optical density values at specific wavelengths in a spectrophotometer.

Both protein and haemin levels showed an initial rapid decrease after engorgement; values did not fall to zero in either case but showed marked fluctuation throughout the study period. These fluctuations combined with the high standard errors of the results, made assessments of the physiological age of individual ticks impossible.

Such fluctuating values, however, suggested the possibility that haem biosynthesis might be taking place, and this was tested by injecting the radioactively-labelled haem precursor [4-14C]δ-amino laevulinic acid into engorged nymphs, immediately following their detachment. Both tick species revealed an incorporation of this compound into their haematin content, although neither incorporated the non-haem precursor [1-14C]2-amino isobutyric acid. These results indicate an ability of ticks to synthesize haem in vivo, although the underlying reasons for such a mechanism remain unknown.  相似文献   


11.
β-Sitosterol-4-14C is metabolized to Δ4-β-sitosten-3-one by Cheiranthus cheiri leaf homogenates. Greater than 60% conversion occurs within 2 hr. Under identical conditions, leaf homogenates of Strophanthus kombé fail to metabolize β-sitosterol, while Digitalis purpurea leaf homogenates yield only very small amounts of the metabolite.  相似文献   

12.
A method is described for measuring (-)-threo-chlorocitric acid in human plasma. Plasma is acidified to pH 1 to minimize lactonization and a13C analogue of (-)-threo-chlorocitric acid is added as internal standard. The acidified plasma is then extracted with ethyl acetate containing 10% methanol. The ethyl acetate—methanol extract is back-extracted with acetate buffer (pH 5). This extract, following adjustment to pH 1, is reextracted with ethyl acetate. The residue after removal of the ethyl acetate is treated with ethereal diazomethane. The wet residue is reconstituted in ethyl acetate and a portion of this solution is analyzed by gas chromatography—chemical ionization mass spectrometry. The mass spectrometer is set to monitorm/z269 [MH+ of trimethylated (-)-threo-chlorocitric acid] andm/z270 [MH+ of trimethylated (-)-threo-[13C]chlorocitric acid] in the gas chromatographic effluent. Them/z/269 tom/z270 ion ratio in a sample containing an unknown amount of (-)-threo-chlorocitric acid is converted to an amount of compound using a calibration curve. The calibration curve is generated by analyzing control plasma spiked with various known amounts of (-)-threo-chlorocitric acid and a fixed amount of (-)-threo-[13C]chlorocitric acid. The limit of quantitation is 0.1–0.6 μg ml−1, depending on the characteristics of the calibration curve generated with each set of samples. The precision (relative standard deviation) at a concentration of 2 μg ml−1 is 3.3%.  相似文献   

13.
The incorporation of [4-14C]4-oxostearic acid, [4-14C]stearic acid, and [1-14C]eicosanoic acid, respectively, into the nonacosan-15-one of Brassica oleracea has been investigated. While the oxo-acid was found to be a poor precursor of nonacosan-15-one, the two n-fatty acids were efficiently incorporated as intact units into n-nonacosane and the related 15-ketone. In accordance with expectation, the label in the 4-position of stearic acid appeared in the carbonyl carbon of the ketone. These results suggest that the C29 hydrocarbon is first formed and subsequently oxidized to nonacosan-15-one.  相似文献   

14.
The addition of gibberellic acid and some other gibberellins to cultures of Gibberella fujikuroi suppresses the incorporation of [2-14C]MVA and 14C-labelled ent-kaurene into the gibberellin metabolises.  相似文献   

15.
Carbon isotope ratios (13C/12C) were measured for the leaves of the seagrass Thalassia testudinum Banks ex König and carbonates of shells collected at the seagrass beds from seven sites along the coast of southern Florida, U.S.A. The δ13C values of seagrass leaves ranged from −7.3 to −16.3‰ among different study sites, with a significantly lower mean value for seagrass leaves from those sites near mangrove forests (−12.8 ± 1.1‰) than those far from mangrove forests (−8.3 ± 0.9‰; P < 0.05). Furthermore, seagrass leaves from a shallow water area had significantly lower δ13C values than those found in a deep water area (P < 0.01). There was no significant variation in δ13C values between young and mature leaves (P = 0.59) or between the tip and base of a leaf blade (P = 0.46). Carbonates of shells also showed a significantly lower mean δ13C value in the mangrove areas (−2.3 ± 0.6‰) than in the non-mangrove areas (0.6 ± 0.3‰; P <0.025). In addition, the δ13C values of seagrass leaves were significantly correlated with those of shell carbonates (δ13C seagrass leaf = −9.1 + 1.3δ13C shell carbonate (R2 = 0.83, P < 0.01)). These results indicated that the input of carbon dioxide from the mineralization of mangrove detritus caused the variation in carbon isotope ratios of seagrass leaves among different sites in this study.  相似文献   

16.
The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-3H2,4-14C]estradiol, 7-ethoxycoumarin, and [N-methyl-3H3]cocaine. 6-Hydroxy[7-3H,4-14C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6-3H label was established. The initial rate kinetics of the 6-hydroxylation gave Km of 4.3 μM, Vmax of 4.02 nmol min−1mg−1, and turnover rate of 0.27 min−1. Testosterone competed dose-dependently with the 6-hydroxylation and showed the Ki of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 μM, Vmax of 12.5 nmol min−1mg−1 and turnover rate of 1.06 min−1. The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 μM, Vmax of 4.76 nmol min−1mg−1, and turnover rate of 0.49 min−1. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min−1mg−1 (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min−1.  相似文献   

17.
Interactions between guanine nucleotide regulatory proteins (G proteins) and muscarinic acetylcholine receptors (mAChRs) were studied in vivo following carbachol treatment. Rat brain homogenates were separated by high speed ultracentrigation into heavy and light membrane and 300,000 g supernate franctions. The G proteins were partially purified by Sephadex-G200 and heptylamine-Sepharose and the mAChRs by (3,2′-aminobenzhydryloxy)-tropane-(ABT)-affinity chromatographies. Radioligand binding assays showed that acute carbachol induced a biphasic translocation of the mAChRs and G proteins into the light membrane fraction with an initial release at 5–10 min and a second phase at 60 min. Portions of the released mAChRs and the G proteins, were found in the 300,000 g supernates and light membranes and were eluted in the same peak fractions from a Sephadex G-200 column. This dually labelled peak dissociated in the presence of digitonin, suggesting close association between the mAChR and G protein. ABT-affinity chromatography yielded dually labelled mAChR-G protein fractions which eluated as a single radioactive peak on a second ABT column. the partially purified G proteins from these fractions were photoaffinity labelled with 8-azidoguanosine-5′-triphosphate, [γ-32P]. SDS-PAGE autoradiography revealed the presence of G and Gi which may be released simultaneously with the mAChRs from the plasma membrane. In addition, a 110,000 molecular weight polypeptide was dually labelled by [3H]-PrBCM and [γ-32P]-8-azido-GTP suggesting the presence of a “mAChR-G protein complex.” These findings provide direct evidence for the release of mAChRs and G proteins and a mAChR-G protein complex by agonist occupation of the mAChRs.  相似文献   

18.
Previous investigations demonstrated that respiratoly reductive dechlorination of vinyl chloride (VC) can be efficient even at H2 concentrations (≤2 nM) that are characteristic of SO4-reducing conditions. In the study reported here, microorganisms indigenous to a lake-bed sediment completely mineralized [1,2-14C] ethene to 1414CO2 when incubated under SO4-reducing conditions. Together, these observations argue for a novel mechanism for the net anaerobic oxidation of VC to CO2: reductive dechlorination of VC to ethene followed by anaerobic oxidation of ethene to CO2. Moreover, the results of this study suggest that reliance on ethene and/or ethane accumulation as a quantitative indicator of complete reductive dechlorination of chioroethene contaminants may not be warranted.  相似文献   

19.
Feeding experiments with 12-phenyl[2,2-2H2]dodecanoic acid and the correponding methylester resulted in the formation of 2-phenylethanol (probably produced via β-oxidation) and high amounts of 2-phenylethylesters of free fatty acids from the defensive secretion of Oxytelus sculpturatus rove beetles. The extraordinarily high amount of the metabolites occurring in the glands after administration of methyl-12-phenyl[2,2-2H2]dodecanoate had a significant effect on the toxicity of the toluquinone-saturated defensive secretion mixture. Analogous experiments with 11-phenyl-[2-2H]undecanoic acid revealed a less efficient incorporation of this precursor leading to esters of 1-phenylethanol and 4-phenylbutan-2-ol and traces of 10-phenyl-1-decene probably formed via oxidative decarboxylation.  相似文献   

20.
In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E1), estradiol (E2), 16-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-3H]testosterone (1.24 × 103 dpm 3H/ng, 35–150 nM) or [1β-3H,4-14C]androstenedione (3.05 × 103 dpm 3H/ng, 3H/14C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E1, E2, 16-OHE1, and E3 for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the Km of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The Ki of E1, E2, 16-OHE1, and E3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the Km of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.  相似文献   

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