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1.
Saito K  Nick JA  Loewus FA 《Plant physiology》1990,94(3):1496-1500
d-[6-14C]Glucosone that had been prepared enzymically from d-[6-14C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, 14C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-14C]glucose underwent considerable redistribution during AA formation, whereas 14C from [6-14C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-14C]sorbosone was found to be equivalent to [6-14C]glucose as a source of 14C for AA. In the presence of 0.1% d-glucosone, conversion of [6-14C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.  相似文献   

2.
The metabolic fate of l-[4-14C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-14C]ascorbic acid and l-[6-14C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-14C]ascorbic acid and l-[1-14C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-14C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-14C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.  相似文献   

3.
Zhu Y  Shearer G  Kohl DH 《Plant physiology》1992,98(3):1020-1028
Supplying l-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding l-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylene-reducing activity. Twenty-four hours after irrigating with l-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-14C]l-proline was supplied to uprooted, intact plants incubated in aerated solution, 14C-labeled products of proline metabolism, as well as [14C]proline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-3H]l-proline, 3H-labeled proline was found in host cells and bacteroids. [3H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-3H]l-proline. When [U-14C]l-proline was supplied for 24 hours, a significant amount of [14C] pyrroline-5-carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-3H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the in vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules.  相似文献   

4.
l-Canavanine, the guanidinooxy structural analog of l-arginine, is an important nonprotein amino acid of many leguminous plants with nitrogen storage a major proported role. l-[Guanidinooxy-14C]canavanine, [14C] urea, and [15N]urea were injected separately into the fleshy, green cotyledons of 9-day old jack bean plants, Canavalia ensiformis (L.) DC. [Leguminosae]. There was significant transport of canavanine from the cotyledons to the aboveground portions of the plant, but not to the roots. Within 1.5 hours of isotope administration, the remaining labeled canavanine was divided equally between the cotyledons and the aboveground portions of the plant. During the 48-hour postinjection period, the contribution of l-[guanidinooxy-14C]canavanine to the total 14carbon of the cotyledons decreased rapidly while it increased in the aboveground portions of the plant.  相似文献   

5.
1. Liver slices from cod (Gadus morhua L.) were incubated with l-[14C]leucine and the incorporation of label into total protein, precipitated with trichloroacetic acid, and into egg proteins, precipitated with an antibody after addition of carrier egg proteins, was measured. 2. Liver slices from immature male or female cod, and from male fish with developing testes, did not incorporate significant amounts of l-[14C]leucine into egg proteins, whereas with slices from female cod with developing ovaries the rate of incorporation into egg proteins was 8% of the rate of incorporation into total protein. 3. Liver slices from immature male or female fish that had received an intramuscular injection of oestradiol benzoate (1mg/kg) 5–8 days previously incorporated l-[14C]leucine into egg proteins at about 26% of the rate of incorporation into total protein. 4. Incorporation into total protein and into egg proteins was inhibited by puromycin, and 1.2 and 0.13μg of puromycin/mg of tissue protein, respectively, gave 50% inhibition.  相似文献   

6.
Sato N 《Plant physiology》1988,86(3):931-934
Biosynthesis of the polar group of diacylglyceryl-O-4′-(N,N,N-trimethyl)homoserine (DGTS) was studied in intact cells of Chlamydomonas reinhardtii Dangeard. Among the three C4 amino acids tested, only l-methionine could specifically inhibit the photosynthetic incorporation of [14C]NaHCO3 into the polar group of DGTS. The radioactivity in l-[14C]methionine, which was labeled at either the C3 + C4, the C1, or the methyl carbon, was efficiently incorporated into the polar group of DGTS. These results suggest that the C4 backbone and the S-methyl group of l-methionine are precursors to the C4 backbone and the N-methyl groups of DGTS, respectively.  相似文献   

7.
Conversion of d-[5-3H,6-14C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Hér cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a 3H:14C ratio of 4 to be compared with 9, the ratio in d-[5-3H,6-14C]glucose initially. Conversion of d-[6-3H,6-14C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a 3H:14C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom.  相似文献   

8.
The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d-glycerate and, usually, [3H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [14C]d-glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [14C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [14C]d-glycerate was strongly inhibited by HgCl2, which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [14C]glycolate accumulation under similar conditions was much greater than that of [14C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [14C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly.  相似文献   

9.
myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-3H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-14C]inositol, myo-[2-3H]inositol, l-[5-14C]arabinose, and d-[5R,5S-3H]xylose.  相似文献   

10.
Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H+ efflux from the cells by 111% and the uptake of l-[U-14C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO2 evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and 14CO2 evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas 14CO2 evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-14C]glutamate were higher in the light while rates of 14CO2 evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO2 and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H+/l-glutamate uptake process.  相似文献   

11.
Degradation of phenylalanine and tyrosine by Sporobolomyces roseus   总被引:3,自引:2,他引:1  
Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-14C]phenylalanine and l-[U-14C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [14C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.  相似文献   

12.
The metabolism of myo-inositol-2-14C, d-glucuronate-1-14C, d-glucuronate-6-14C, and l-methionine-methyl-14C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-14C contained labeled polysaccharide.  相似文献   

13.
The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. l-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with l-[U-14C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular l-Glu metabolites. Incubation with l-[1-14C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with l-[U-14C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 × 10−1 nanocuries per nanomole. In the presence of exogenous l-Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that l-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA.  相似文献   

14.
1. all-trans-Retinoic acid at concentrations greater than 10−7m stimulated the incorporation of d-[3H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl2-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of 35SO42−, l-[14C]fucose and U-14C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[3H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10−5m-all-trans-retinoic acid. The labelling of other epidermal components with d-[3H]glucosamine, 35SO42−, l-[14C]fucose and U-14C-labelled l-amino acids was not significantly affected by 10−5m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[3H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10−5m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[3H]-glucosamine into extracellular epidermal glycosaminoglycans in 10−5m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10−5m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10−5m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[3H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10−5m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.  相似文献   

15.
Evidence for a specific glutamate/h cotransport in isolated mesophyll cells   总被引:1,自引:1,他引:0  
Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO4. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H+/106 cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-14C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K+ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.  相似文献   

16.
Others have shown that l-methionine is utilized in the biosynthesis of methyl ester groups in pectic substance. Methanol, like l-methionine, is used for methyl ester biosynthesis by detached parsley leaves (Petroselinum crispum). When a combination of methanol-3H and methanol-14C is given to parsley leaves, methanol recovered from pectic substance by alkaline hydrolysis has a 3H/14C ratio about one-fourth that of the mixture administered. Unlike l-methionine, methanol is oxidized prior to its utilization as a carbon source for methyl ester biosynthesis.  相似文献   

17.
Tracerkinetic experiments were performed using l-[guanidino-14C]arginine, l-[U-14C]arginine, l-[ureido-14C]citrulline, and l-[1-14C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free 14C compounds and incubated in sealed vials containing a CO2 trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the 14C metabolized from [guanidino-14C]arginine and [U-14C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours 14CO2 was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra 14CO2 was evolved during the catabolism of ornithine-derived glutamate; 14C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.  相似文献   

18.
Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two Ambrosiozyma monospora transporters were successfully expressed in Saccharomyces cerevisiae as tagged proteins. Growth of A. monospora on l-arabinose instead of glucose caused transport activities of l-arabinose, l-arabitol, and ribitol, measured using l-[1-3H]arabinose, l-[14C]arabitol, and [14C]ribitol of demonstrated purity. A. monospora LAT1 and LAT2 genes were cloned earlier by using their ability to improve the growth of genetically engineered Saccharomyces cerevisiae on l-arabinose. However, the l-arabinose and pentitol transport activities of S. cerevisiae carrying LAT1 or LAT2 are only slightly greater than those of control strains. S. cerevisiae carrying the LAT1 or LAT2 gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for LAT1; >20-fold for LAT2) increases in transport activities. Lat1-mCherry transported l-arabinose with high affinity (Km ≈ 0.03 mM) and l-arabitol and ribitol with very low affinity (Km ≥ 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport l-arabinose but were high-affinity pentitol transporters (Kms ≈ 0.2 mM). The l-arabinose and pentitol transport activities of A. monospora could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that A. monospora contains at least one more l-arabinose transporter.  相似文献   

19.
Chisholm MD  Wetter LR 《Plant physiology》1967,42(12):1726-1730
The incorporation of the radioactivity from acetate-1-14C, acetate-2-14C, dl-methionine-1-14C, dl-methionine-2-14C, dl-methionine-3,4-14C, dl-homomethionine-2-14C, dl-allyl-glycine-2-14C, and dl-2-amino-5-hydroxyvalerate-2-14C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-14C, dl-methionine-3,4-14C, dl-homomethionine-2-14C, and acetate-2-14C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-14C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.  相似文献   

20.
We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[15N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[15N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.  相似文献   

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