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1.
The contents of free sugars in nodules of chickpea (Cicer arietinum) were maximum around flowering. In stem and root tissues, the relative incorporation of 14C from [ 14C]-labelled sucrose or glucose into extracted sucrose was over 70 %. In the former tissue, the relative incorporation of 14C from glutamate into sucrose was about 50 % at 50 d after sowing (DAS) but the same decreased to about 25 % at 80 DAS. However, from glutamate, 63–68 % of 14C from extracted sugars of root tissue appeared in invert sugars. Feeding via stem [ 14C]-glutamate to intact nodules led to intense labelling of sucrose and invert sugars in nodule cytosol. Upon injecting labelled sugars or glutamate into isolated nodules, maximum 14C appeared in glucose of this nodule fraction. In bacteroids, incorporation of 14C from glutamate was much higher in amino acids. In the cytosol of younger (50 DAS) nodules, sucrose was cleaved largely by soluble alkaline invertase (EC 3.2.1.26). However, sucrose cleavage in this fraction of older (80 DAS) nodules was catalysed by this enzyme as well as sucrose synthase (reversal, EC 2.4.1.13) and such nodules also contained higher activity of nitrogenase. The bacteroid fraction, which contained 10–17 % of nodule sugars, lacked the activities of sucrose-cleaving enzymes. The activities of ATP-dependent phosphofructokinase (EC 2.7.1.11), glyceraldehyde-3-phosphate dehydrogenase (EC 1.1.1.12), NADP +-dependent isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) were higher in cytosol than bacteroids. However, the reverse was true for glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The results suggest that in chickpea nodules sugar metabolism occurs largely via the glycolytic pathway in cytosol and the pentose phosphate pathway in bacteroids and there is some transport of glutamate from cytosol to bacteroids. 相似文献
2.
Labeling studies using detached lupin ( Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4- 14C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1- 14C]- and [6- 14C]glucose, while with [U- 14C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction. 相似文献
3.
The metabolism of translocated photosynthate by soybean ( Glycine max L. Merr.) nodules was investigated by 14CO 2-labeling studies and analysis of nodule enzymes. Plants were exposed to 14CO 2 for 30 minutes, followed by 12CO 2 for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate). The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction. In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in α,α-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids. 相似文献
4.
In nodules of Glycine max cv. Mandarin infected with a nod +fix - mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod + fix - mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean. 相似文献
5.
On feeding 14CO 2 to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of 14C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids. 相似文献
6.
Abstract— Rat brain cortex slices were incubated with 10 mm -glucose and trace amounts of [6- 3H]glucose and [3- 14C] β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and 3H: 14C ratios calculated. Incorporation of [ 14C] β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of 14C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [ 3H-, 14C]-glucose. (–)-Hydroxycitrate (5.0 m M) reduced incorporation of [ 3H]glucose and [ 14C] β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm ) showed a decrease in 14C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm ) also produced a decreased 14C incorporation into ACh and its precursors. At 10 mm , acetoacetate reduced 3H and 14C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced. 相似文献
7.
The aim of this work was to discover how leucoplasts from suspension cultures of soybean ( Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with 14C-labelled substrates led to 14CO 2 production, that was dependent upon leucoplast intactness, from [U- 14C]glucose 6-phosphate, [U- 14C]glucose 1-phosphate, [U- 14C] fructose 6-phosphate and [U- 14C]glucose+ATP, but not from [U- 14C]fructose-1,6-bisphosphate or [U- 14C]triose phosphate. The yield from [U- 14C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1- 14C]-, [2- 14C]-, [3,4- 14C]-, and [6- 14C]glucose 6-phosphate were supplied to leucoplasts significant 14CO 2 production that was dependent upon leucoplast intactness was found only for [1- 14C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship. 相似文献
8.
Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of 14C into triterpenes from [2- 14C]mevalonic acid, [1- 14C]acetate, [3- 14C]pyruvate, [U- 14C]glyoxylate, [U- 14C]glycerol, [U- 14C]serine, [U- 14C]xylose, [U- 14C]glucose, and [U- 14C]sucrose was obtained. The [ 14] triterpenes synthesized from [ 14C] sugars were mainly of latex origin. [ 14C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The 14C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [ 14C]sugars, [ 14C]acetate, and [ 14C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [ 14C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings. 相似文献
9.
Intact caeca of the marine borer, Bankia setacea (Tryon), were incubated in vitro with (1- 14C)- and (6- 14C)-glucose. The specific yields of 14CO 2 from (1- 14C)- and (6- 14C)-glucose were found to be 9 and 1% respectively. From these values the contribution of the pentose cycle to the overall glucose metabolism was calculated as 3%. Glucose is catabolized mainly via the Embden-Meyerhof pathway. 相似文献
10.
l-Ascorbic acid (AA) production in cells of Cyclotella cryptica Reimann, Lewin, Guillard (Bacillariophyceae) is enhanced when darkadapted cells are exposed to light.Heterotrophically grown cells incubated with d-[6- 3H,6- 14C]glucose and d-[1- 3H,6- 14C]glucose (2 h in dark followed by 15 h light) produced labeled AA with significantly different ratios of 3H and 14C. Comparisons of labeling patterns in AA and chitin-derived d-glucosamine support a path of conversion in Cyclotella from d-glucose to AA that inverts the carbon chain of the sugar. This process resembles similar conversions found in AA-synthesizing animals and species from two other algal classes.Abbreviations AA
l-Ascorbic acid
- glc
d-glucose
- glcN
d-glucosamine 相似文献
11.
Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U- 14C]-glucose utilisation was calculated by summing the 14C found in CO 2, triglycerides, and anions. [ 3H]-H 2O formation from [5- 3H]-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the [5- 3H]-glucose is simpler to use than [U- 14C]-glucose and gives substantially more reproducible results than glucose oxidase. 相似文献
12.
We studied the effect of different concentrations of 2-deoxy- d-glucose on the l-[U- 14C]leucine, l-[1- 14C]leucine and [1- 14C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy- d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U- 14C]leucine and from [1- 14C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy- d-glucose inhibited the CO 2 production and lipid synthesis from l-[U- 14C] leucine. This compound did not inhibit either CO 2 production, or lipid synthesis from [1- 14C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy- d-glucose on the protein synthesis from l-[U- 14C]leucine. 2-deoxy- d-glucose at 2.0 mM did not show any effect either on CO 2 production, or on lipid synthesis from l-[U- 14C]lactate 10 mM and glucose 5.0 mM. 相似文献
13.
Rhizodeposition, i.e. the release of carbon into the soil by growing roots, is an important part of the terrestrial carbon
cycle. However the in situ nature and dynamics of root-derived carbon in the soil are still poorly understood. Here we made an investigation of the
latter in laboratory experiments using 13CO 2 pulse chase labelling of wheat ( Triticum aestivum L.). We analyzed the kinetics of 13C-labelled carbon and more specially 13C carbohydrates in the rhizosphere. Wheat seedlings-soil mesocosms were exposed to 13CO 2 for 5 hours in controlled chambers and sampled repeatedly during two weeks for 13C/C analysis of organic carbon. After a two-step separation of the soil from the roots, the amount of total organic 13C was determined by isotope ratio mass spectrometry as well as the amounts of 13C in arabinose, fructose, fucose, glucose, galactose, mannose, rhamnose and xylose. The amount and isotopic ratio of monosaccharides
were obtained by capillary gas chromatography coupled with isotope ratio mass spectrometry (GC/C/IRMS) after trimethyl-silyl
derivatization. Two fractions were analyzed : total (hydrolysable) and soluble monomeric (water extractable) soil sugars.
The amount of organic 13C found in the soil, expressed as a percentage of the total photosynthetically fixed 13C at the end of the labelling period, reached 16% in the day following labelling and stabilised at 9% after one week. We concluded
that glucose under the form of polymers was the dominant moietie of rhizodeposits. Soluble glucose and fructose were also
present. But after 2 days, these soluble sugars had disappeared. Forty percent of the root-derived carbon was in the form
of neutral sugars, and exhibited a time-increasing signature of microbial sugars. The composition of rhizospheric sugars rapidly
tended towards that of bulk soil organic matter. 相似文献
14.
Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with 14C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O 2 (2% in the gas phase). Uptake and conversion of 14C to CO 2 were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO 2, and fructose was respired at a rate at least double that of glucose. Sucrose and glucose were converted to CO 2 at a very low but measurable rate (<0.1 nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO 2 at a rate 30 times greater than the conversion of carbon Number 6 to CO 2, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase. 相似文献
15.
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
16.
High resolution 13C NMR combined with chemical analysis were used to study the formation of metabolites from [1- 13C]-labelled glucose by the salt-tolerant yeast Debaryomyces hansenii after transfer to media containing 8% NaCl. Time course spectroscopy of an aerobic cell suspension showed [1,3- 13C]glycerol as the predominant end product. Perchloric acid extracts revealed additional less prominent incorporation of label into arabinitol, trehalose, glutamic acid, and alanine. The incorporation into trehalose and arabinitol showed a transient increase after shift to the high salinity medium. It is concluded that glycerol and arabinitol are the major organic solutes in D. hansenii, the production of glycerol being strongly induced by high salinity. Analysis of labelled extracts of D. hansenii after transfer to 8% NaCl media containing [1- 13C]- or [6- 13C]glucose, demonstrated that glucose is dissimilated via a combination of the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway, with the former playing a major role in glycerol formation and the latter in arabinitol production. The almost exclusive labelling of C5 of arabinitol from [6- 13C]glucose indicates that the pathway to arabinitol proceeds via reduction of ribulose-5-phosphate.Abbreviations used NMR
nuclear magnetic resonance
- EMP
Emden-Meyerhof-Parnas
- PP
pentose phosphate
- GAP
glyceraldehyde phosphate
- DHAP
dihydroxyacetone phosphate
- ppm
parts per million 相似文献
18.
Nitrate, nitrite and nitrous oxide were denitrified to N 2 gas by washed cells of Rhizobium japonicum CC706 as well as by bacteroids prepared from root nodules of Glycine max (L.) Merr. (CV. Clark 63). Radiolabelled N 2 was produced from either K 15NO 3 or Na 15NO 2 by washed cells of Rh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N 2O. Similar results were obtained with bacteroids of G. max. The stoichiometry for the utilization of 15NO
3
-
or 15NO
2
-
and the produciton of 15N 2 was 2:1 and for N 2O utilization and N 2 production it was 1:1. Some of the 15N 2 gas produced by denitrification of 15NO
3
-
in bacteroids was recycled via nitrogenase into cell nitrogen. 相似文献
19.
The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na +/ myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential ( V
m
), the external myo-inositol concentration and the external Na + concentration, yielding the kinetic parameters: K
0.5
MI
, K
0.5
Na
, and the Hill coefficient n. At 100 mM NaCl, K
0.5
MI
was about 50 m and was independent of V
m
. At 0.5 m m
myo-inositol, K
0.5
Na
ranged from 76 m m at V
m
=–50 mV to 40 m m at V
m
=–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na + ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K
I
of 64 m at V
m
=–50 mV and 130 m at V
m
= –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V
m
=–150 mV) were recorded for a series of sugars, each at an external concentration of 50 m m. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl- d-glucopyranoside > d-galactose, d-fucose, 3- O-methyl- d-glucose, 2-deoxy- d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na +/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V
m
=–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl- d-glucopyranoside, d-galactose, d-fucose, 3- O-methyl- d-glucose > d-xylose, l-xylose, 2-deoxy- d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na +-dependent uptake of 3H- d-glucose and 3H- myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 m m Na + that was abolished by phlorizin (0.5 m m). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V
m
. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.
Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812
Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554. 相似文献
20.
A labeling scheme is introduced that facilitates the measurement of accurate 13C β chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use
of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels
of 13C enrichment (30–40%) at C β side-chain carbon positions for 15 of the amino acids with little 13C label at positions one bond removed (≈5%). A pair of samples are produced using [1- 13C]-glucose/NaH 12CO 3 or [2- 13C]-glucose as carbon sources with isolated and enriched (>30%) 13C β positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy.
The utility of such samples for measurement of 13C β chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion
studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles
compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples. 相似文献
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