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1.
Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K+, Mg2+-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent Km (ATP) and Vmax of 0.75 m M and 79 μmol (mg protein)−1 h−1, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 m M and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.  相似文献   

2.
Nickel and rubidium uptake by whole oat plants in solution culture   总被引:1,自引:0,他引:1  
Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni2+ and Rb+ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni2+ and Rb+ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni2+ uptake by 93%. Nickel and Rb+ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. Km (0.012 m M ) and Vmax [2.72 μmol (g dry weight)-1 h-1] values for Ni2+ were nearly 7 times lower than those for Rb+ [0.09 m M and 19.2 μmol (g dry weight)-1 h-1]. In all experiments, Ni2+ and Rb+ showed qualitatively similar uptake patterns, but Rb+ uptake was quantitatively more sensitive than Ni2+ to experimental manipulations.  相似文献   

3.
Iron inefficiency in the maize ( Zea mays L.) mutant ysl is caused by a defect in the uptake system for Fe-phytosiderophores. To characterize this defect further, the uptake kinetics of Fe-phytosiderophores in ysl was compared to the Fe-efficient maize cultivar Alice. Short-term uptake of 59Fe-labeled Fe-deoxymugineic acid (Fe-DMA) was measured over a concentration range of 0.03 to 300 μM. Iron uptake in Fe-deficient plants followed Michaelis-Menten kinetics up to about 30 μM and was linear at higher concentrations, indicating two kinetically distinct components in the uptake of Fe-phytosiderophores. The saturable component had similar Km (∼ 10 μM) in both genotypes. In contrast. Vmax was 5.5 μmol Fe-DMA g−1 dry weight [30 min]−1 in Alice, but only 0.6 μmol Fe-DMA g−1 dry weight [30 min]−1 in ysl. Uptake experiments with double-labeled 59Fe-[14C]DMA suggest that in both cultivars Fe-DMA was taken up by the roots as the intact chelate. The results indicate the existence of a high-affinity and a low-affinity uptake system mediating Fe-phytosiderophore transport across the root plasma membrane in maize. Apparently, the mutation responsible for Fe inefficiency in ysl affected high-affected uptake and led to a decrease in activity and/or number of Fe-phytosiderophore transporters.  相似文献   

4.
Abstract— When suboesophageal ganglia of the snail Helix comalia were incubated at 25°C in a medium containing [3H]choline, tissue: medium ratios of about 14:1 were obtained after 20 min incubation, and only 15°, of the accumulated choline was metabolized to form [3H]acetylcholine. The uptake of [3H]choline showed saturation kinetics and was dependent upon temperature and sodium ions. Kinetic analysis suggested the existence of a high affinity uptake process (Km= 1.7 μM, Vmax= 0.21 nmol/g/min) and a low affinity process (Km= 100 μM, Vmax= 1.2 nmol/g/min). The high affinity uptake differed from the low affinity system in that it was sensitive to various metabolic inhibitors and was competitively inhibited by low concentrations of hemicholinium- and acetylcholine. Neither uptake system was greatly influenced by the absence of calcium, potassium or magnesium ions or by the presence of low concentrations of 5-HT, dopamine. tetrabenazine, chlorpromazine, decamethonium, nalaxone or imipramine. The high affinity uptake process may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.  相似文献   

5.
The synthesis of homoglutathione (hGSH) by several plants of the tribe Phaseoleae is shown to be catalysed by a β-alanine-specific hGSH synthetase, Properties of the enzyme from Phaseolus coccineus L. cv. Preisgewinner were studied, using ammonium sulfate precipitates of primary leaf extracts. The hGSH synthetase showed a broad pH optimum at pH 8–9, an absolute requirement for Mg2+, a stimulation by K+, and a high affinity for γ-glutamylcysteine [Km(app.) 73 μ M ]. The enzyme exhibited a high specificity for β-alanine [Km(app.) 1.34 m M ] compared to glycine [Km(app.) 98 m M ]. Chloroplasts, isolated from the leaves of Phaseolus coccineus , contained about 17% of the hGSH synthetase activity in the leaf cells.  相似文献   

6.
The effects of copper (CuCl2) on active and passive Rb+(86Rb+) influx in roots of winter wheat grown in water culture for 1 week were studied. External copper concentrations in the range of 10–500 μ M in the uptake nutrient solution reduced active Rb+ influx by 20–70%, while passive influx was unaffected (ca 10% of the Rb+ influx in the Cu-free solution). At external Rb+ concentrations of up to 1 m M , Cu exposure (50 μ M decreased Vmax to less than half and increased Km to twice the value of the control. Short Cu exposure reduced the K+ concentration in roots of low K+ status. Pretreatment for 5 min in 50 μ M CuCl2 prior to uptake experiments reduced Rb+ influx by 26%. After 60 min pretreatment with Cu, the corresponding reduction was 63%. Cu in the cultivation solution impeded growth, especially of the roots. The Cu concentration in the roots increased linearly with external Cu concentration (0–100 μ M ) while Cu concentration in the shoots was relatively unchanged. The K+ concentration in both roots and shoots decreased significantly with increased Cu in the cultivation solutions. Possible effects of Cu on membranes and ion transport mechanisms are discussed.  相似文献   

7.
Nitrogen (N) deficiencies in tundra ecosystems could be caused, in part, by the kinetics of root N uptake. The objectives of this study were to quantify NH4 uptake by field-grown excised roots of Eriophorum vaginatum I. under controlled NH4 concentrations (0-250 μmol I-1) and temperatures (5-20°C) and to evaluate this laboratory derived model as a means of estimating field NH4 uptake. There was no consistent temperature effect on root NH4 uptake which suggests a relative in-sensitivity of E. vaginatum roots to short-term temperature fluctuations. The Michaelis-Menten equation parameters for NH4 uptake were Vmax= 22.1 μmol h-1 g-1 and Km= 191 μmol I-1. Using field NH4 concentrations, field E. vaginatum root biomass data, and the Michaelis-Menten equation, an estimate was made of NH4 uptake over a 42 day period; this estimate of NH4 uptake accounted for 28% of the net incorporation of N into leaves and roots which is a reasonable estimate for E. vaginatum which relies primarily on N retranslocation for supplying new leaves and roots. Major uncertainties in field N uptake rates, model parameterization, and site characterization preclude an accurate model validation and indicate research areas most in need of future study.  相似文献   

8.
Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a Km, of 28.8 ± 2.8 μM and a Vmax of 0.294 ± 0.022 nmol × min−1× [mg cell protein]−1. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC50) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.  相似文献   

9.
Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH4)2SO4 precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)-1 min-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn2+ or Mg2+). In the presence of MnCl2+ other divalent cations (Mg2+, Ca2+, Ba2+, Hg2+ and Cd2+) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The Km for adenine was 4.5±1.5 μ M , the Km for PRPP was 0.29±0.06 m M and the Ki for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The Km for benzyladenine was approximately 0.73±0.06 m M  相似文献   

10.
NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)−1 min−1 at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH2-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent Km for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent Km for nitrate as well as Vmax whereas it did not alter the Km for NADH.  相似文献   

11.
Abstract: Recent studies indicate the lumped constant (LC), which defines the relative rates of brain utilization of glucose and 2-deoxyglucose (2-DG), doubles to values > 1.0 under conditions of hypoglycemia. Since changes in the LC should be predictable given the kinetic parameters of blood-brain barrier (BBB) transport and brain phosphorylation of glucose and 2-DG, the present studies were designed to measure the necessary kinetic parameters. The carotid injection technique was used to determine cerebral blood flow and the Km , Vmax, and K D of glucose and 2-DG transport through the BBB in seven brain regions in rats anesthetized with 50 mg/kg i.p. pentobarbital. Regional glucose transport through the BBB was characterized by an average Km = 6.3 m m , average Vmax = 0.53 μmol min−1g−1, and average K D= 0.022 ml min−1g−1. The nonsaturable route of transport of glucose represented on the average 40% of the total glucose influx into brain regions at an arterial glucose concentration of 10 m m . In addition, the rate constants of phosphorylation of glucose and 2-DG were measured for each region. Substitutions of the measured kinetic parameters for sugar transport and phosphorylation into equations defining the LC confirm the observation that the LC would be expected to vary under extreme conditions such as hypoglycemia and to exceed values of 1.0 under these conditions.  相似文献   

12.
β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The Km and Vmax values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)-1, h-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co24, Cu2+, Hg2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.  相似文献   

13.
Atlantic salmon ( Salmo salar L.) fry hatched from eggs transferred from high-Na to low-Na water during the eyed stage of development had a significantly higher Vmax and lower Km (P <0.01) of the sodium uptake mechanism than fry hatched from eggs incubated entirely in low-Na or high-Na water.
Fry hatched from eggs transferred to acid, high aluminium water during the eyed stage of development had a similar Vmax and Km to fry hatched from eggs incubated entirely in high- or low-Na water. Eggs incubated continuously in acid, high aluminium (low-Na) water produced fry with significantly lower Km and Vmax values than fry hatched from eggs incubated continuously in low-Na water. Eggs and fry in acid, high aluminium water continually lost sodium and mortality was 100% at 5 5 M O degree-days (2–3 weeks after hatching).
The results are discussed with respect to the influence of perivitelline fluid ion activities in eggs in acid, high aluminium water on the kinetic characteristics of sodium uptake in yolk-sac fry. A possible mechanism for the long-term adaptation of teleosts in acidified natural waters is also proposed.  相似文献   

14.
Changes in sugar uptake into strawberry fruits with maturation and the hormonal effect on uptake mechanisms, though important to fruit development, are not known. Therefore, the kinetics of sugar uptake into strawberry ( Fragaria x ananassa Duch cv. Nyoho) fruit tissue and the effects of abscisic acid (ABA) and indoleacetic acid (LAA) on the mechanism of uptake were investigated at 25 and 35 days after pollination (DAP). Uptake of 14C-sugar was measured over the concentration range of 2 to 30 m M. Uptake kinetics showed a biphasic response to increasing external concentration of 14C-sugars, and indicated the presence of P -chlorormercuribenzenesulfonic acid (PCMBS)-sensitive and PCMBS-insensitive uptake. The Km value for each sugar was in the range of 10 to 20 m M. Stage of development had no effect on Km. but Vmax for glucose decreased with maturation. Further, sucrose was not taken up through a PC-MBS-sensitive transport at 35 DAP. ABA, especially 10 μ M , at 25 DAP stimulated uptake of all sugars, mostly through enhanced PCMBS-insensitive uptake but not PC-MBS-sensitive uptake. In contrast to ABA, stimulation of sugar uptake by IAA was most effective at 1 μ M . The PCMBS-insensitive uptake of each sugar was also stimulated by IAA. Further, the PCMBS-sensitive uptake of glucose was enhanced. The developmental change of PCMBS-sensitive sugar uptake and the effect of ABA and IAA on uptake mechanism in this study are considered to be important in influencing the development and enlargement of fruits.  相似文献   

15.
Ethylene production from an embryogenic culture of Norway spruce ( Picea abies L.) was generally low. ca 2.5 nl g−1 h−1, whereas 1-aminocyclopropane-1 -carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g−1 during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O2) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 μ M ) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 μl I−1) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2.4-dichloro-phenoxyacetic acid (2.4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g−1 h−1 within the 11-day incubation period. Although 2.4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g−1, was observed in tissue treated with 2.4-D (22.5 μ M ) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent Km was 50 μ M and Vmax 2.7 nl g−1 h−1. Growth of the tissue was strongly inhibited by 2.4-D in the absence of BA, but weakly in the presence of BA (4.4 μ M ). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.  相似文献   

16.
Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The Km and Vmax values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)−1 h−1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba2+, Cu2+, Mn2+ and Hg2+. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.  相似文献   

17.
Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H2 ranged from 0.04 to 5.0 μmol H2 (g fresh weight nodule)−1 h−1. The H2 uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed Km for H2 of 13.0 and 23.6 μM H2, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.  相似文献   

18.
Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min−1. mg−1 cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a Km of 220 μ m and a V max of 7.9 nmol. min−1. mg−1 cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca2+-independent had a Km for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.  相似文献   

19.
Chloroplast glutathione reductase: Purification and properties   总被引:4,自引:0,他引:4  
Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)−1 min−1. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The Km for oxidized glutathione was 11 μ M and the Km for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H2O2 by reducing dehydroascorbate for ascorbate-linked reduction of H2O2. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.  相似文献   

20.
The kinetics of ammonium assimilation were investigated in two seaweeds from northeastern New Zealand, Enteromorpha sp. (Chlorophyceae, Ulvales) and Osmundaria colensoi (Hook. f. et Harvey) R.E. Norris (Rhodophyceae, Ceramiales), with the use of a recently developed method for measuring assimilation. In contrast to ammonium uptake, which was nonsaturable, ammonium assimilation exhibited Michaelis–Menten kinetics in both species. Maximum rates of assimilation (Vmax) were 27 and 12 μmol·(g DW)−1·h−1 for Enteromorpha sp. and O. colensoi, respectively, with half-saturation (Km) constants for assimilation of 18 and 41 μM. At environmentally relevant concentrations, assimilation accounted for all of the ammonium taken up by both species. The maximum rate of assimilation in Enteromorpha sp. resembled very closely that of the ammonium assimilatory enzyme, glutamine synthetase, when activities of the latter were measured in the presence of subsaturating substrate (glutamate and ATP) concentrations. Moreover, the initial rate of glutamine production (measured with HPLC) following ammonium enrichment was almost identical to the rates determined above. The rate of ammonium assimilation was therefore determined by three independent methods, two of which involve in vivo measurements, and it is suggested that the use of assimilation kinetics may be useful when examining the nutrient relations of seaweeds.  相似文献   

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