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1.
Abstract Whole cells of the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120 exhibited K m values for l -glutamine and l -glutamate of 33 μM and 0.5 mM, respectively. V max of uptake was ca. 30 nmol mg−1 (chlorophyll) min−1 for both amino acids. The similar pattern of sensitivity to other amino acids exhibited by both transport activities suggests that a common transport system is involved in glutamine and glutamate uptake by this cyanobacterium.  相似文献   

2.
The Uptake of Carnitine by Slices of Rat Cerebral Cortex   总被引:5,自引:3,他引:2  
Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 ± 3 for L-[14CH3]carnitine and 27 ± 3 for D-[14CH3]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of K m for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of V max (1 μmol/min/ml in-tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by γ-butyrobetaine ( K i= 3.22 mM), acetylcarnitine ( K i= 6.36 mM), and γ-aminobutyric acid ( K i= 0.63 mM). The data suggest that carnitine and γ-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N2 atmosphere, 2,4-dinitrophenol, carbonylcyanide- N -chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na+-dependent, but did not require K+ or Ca2+.  相似文献   

3.
Abstract The uptake and incorporation of 75[Se]selenite by Butyrivibrio fibrisolvens and Bacteroides ruminicola were by constitutive systems. Rates of uptake were higher in chemostat culture than in batch culture and there may be some inducible component. Uptake of [75Se]selenite was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit selenite uptake, nor could sulphate or selenate uptake be demonstrated in these organisms. Selenite uptake in B. fibrisolvens had and apparent K m of 1.74 mM and a V max of 109 ng Se · min−1· (mg protein)−1. An apparent K m of 1.76 mM and V max of 1.5 μg Se · min−1· (mg protein)−1 was obtained for B. ruminicola . [75Se]Selenite uptake by both organisms was partially sensitive to inhibition by 2,4-DNP. Uptake by B. fibrisolvens was also partially inhibited by azide and arsenate and in B. ruminicola it was partially inhibited by fluoride. CCCP, CPZ, DCCD or quinine did not inhibit uptake in either B. fibrisolvens or B. ruminicola . Selenite transport by both organisms was sensitive to IAA and NEM and was strongly inhibited by sulphite and nitrite. [75Se]Selenite was converted to selenocystine, selenohomocystine and selenomethionine by B. fibrisolvens. B. ruminicola did not incorporate [75Se]selenite into organic compounds, but did reduce it to red elemental selenium.  相似文献   

4.
5.
Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na+ gradient ([Na+] outside > [Na+] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na+ and Cl ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m -chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na++ K+)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing K m and V max values of similar order to those found in slightly "cruder" preparations. Inhibition of the l -aspartate uptake by d -aspartate and d - and l -glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d - and l -glutamate stereoisomers.  相似文献   

6.
Abstract: Data are presented in support of the transport of (-)- d -3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were Vmax 2.0 μmol.g−1.min−1, K m 29 m M , and K D 0.024 ml.g−1.min−1. The value for Vmax was the same as that for l -lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-0x0-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The K m values for the 2-oxo acids were severalfold lower than the respective K m values. 2-oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.  相似文献   

7.
Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T3) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na+-independent, and efficiently inhibited by T3 (half-inhibition at ∼ 2 μ M ). Two Na+-independent L-like systems (L1 and L2), common to leucine and aromatic amino acids, were characterized kinetically. System L2 had a low affinity for leucine and tryptophan ( K m= 0.3–0.9 m M ). The high-affinity system L1 ( K m∼ 10 μ M for both amino acids) was competitively inhibited by T3 with a K i of 2–3 μ M (close to the T3 transport K m). Several T3 analogues inhibited system L1 and the T3 transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T3 had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T3 transport system and system L1 are related.  相似文献   

8.
The kinetics of active sodium uptake in dechorionated embryos, yolk-sac fry and start-feed fry of Atlantic salmon were compared in two groups reared either in low conductivity, untreated, river water (conductivity ∼ 46 μS cm−1, pH 5.75), or in 'improved' river water buffered with sea water (conductivity ∼2200 μS cm 1, pH 6.56), the latter treatment often being used in commercial hatcheries to avoid problems associated with periodic acidification.
Maximal transport rate ( V max) increased during development in both groups but was always significantly higher in embryos and fry maintained in untreated river water. Values for K m were not seen to vary during development up to 12 weeks after hatching and were not significantly different between groups, or from values reported for adult Atlantic salmon in fresh water.
The results are discussed with respect to the influence of Na+ concentrations in the perivitelline fluid of developing eggs and in the external medium surrounding fry on V max and K m. The ability of fry reared entirely in buffered river water to maintain sodium balance following transfer to untreated river water is also considered.  相似文献   

9.
Abstract A glycerol:NADP+ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K m= 0.01 mM) or NADP+ ( K m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.  相似文献   

10.
Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age-groups. Data were analysed in terms of the Michaelis-Menten equation with a component to allow for non-saturable diffusion. Values are given for K m, V max and K d. In suckling rats (15-21 days) the V max values for both pyruvate and lactate were 2.0 μmol g−1 min−1. In 28-day-old rats the V max values had fallen to one-half and in adults they were less than one-tenth. K m, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28-day-old and adults although there was no difference with age in the K m values.
The results are discussed in relation to the net flux of these substrates in and out of brain during different stages of post-natal development.  相似文献   

11.
High- and Low-Affinity Transport of D-Glucose from Blood to Brain   总被引:21,自引:19,他引:2  
Abstract: Measurements of the unidirectional blood-brain glucose flux in rat were incompatible with a single set of kinetic constants for transendothelial transport. At least two transfer mechanisms were present: a high-affinity, low-capacity system, and a low-affinity, high-capacity system. The low-affinity system did not represent passive diffusion because it distinguished between D-and L-glucose. The Tmax and K m, for the high-affinity system were 0.16 mmol 100 g−1 min−1 and 1 mM; for the low-affinity system, ∼ 5 mmol 100 g−1 min−1 and ∼ 1 M. With these values, physiological glucose concentrations were not sufficient to saturate the low-affinity system. In normoglycemia, therefore, three independent pathways of glucose transport from blood to brain appear to exist: a high-affinity facilitated diffusion pathway of apparent permeability 235·10−7 cm s−1, a specific but nonsaturable diffusion pathway of permeability 85·10−7 cm s−l, and a nonspecifc passive diffusion pathway of permeability 2·10−7 cm s−1.  相似文献   

12.
Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N G-methyl- l -arginine and N G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N G-methyl- l -arginine was characterized by biphasic kinetics ( K m1 = 8 µmol/L, V max1 = 0.09 nmol × mg−1× min−1; K m2 = 229 µmol/L, V max2 = 2.9 nmol × mg−1× min−1) and was inhibited by basic but not by neutral amino acids. Uptake of N G-nitro- l -arginine followed Michaelis-Menten kinetics ( K m = 265 µmol/L, V max = 12.8 ± 0.86 nmol × mg−1× min−1) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N G-methyl- l -arginine is mediated by system y+, whereas systems L and T account for the transport of N G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.  相似文献   

13.
Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K m of 0.20 ± 0.03 mM and 0.28 ± 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V max with a S 0.5 of 15 μM, and no changes in the K m for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.  相似文献   

14.
Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O -acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K' m= 2.16 ± 0.13 μ m and V' max= 27.0 ± 2.9 pmol min−1 mg−1, whereas the low-affinity system has K' m= 20.4 ± 1.3 μ m and V' max= 402 ± 49 pmol min−1 mg−1. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K' m of the high-affinity choline transport system was reduced by 40–60%. The V' max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.  相似文献   

15.
D-GALACTOSE TRANSPORT BY SYNAPTOSOMES ISOLATED FROM RAT BRAIN   总被引:5,自引:3,他引:2  
Abstract— Synaptosomes prepared by differential and Ficoll density gradient centrifugation took up d -galactose by two saturable transport systems: one. a high affinity system with a K m of 0-25 mn and Vmax of 075 nmol/mg protein 3 min, the other, a low affinity system with a Km of 47 mM and a Vmax of 135 nmol/mg protein/3 min. The high affinity system was inhibited by 1-5 mM phlorizin but was unaffected by the absence of sodium ion or the presence of 1 mM ouabain. The low affinity system was unaffected by phlorizin or ouabain. Both systems were inhibited by high concentrations of glucose. 2-deoxyga-lactose. and inositol, and by 2.4-dinitrophcnol. Galactose was rapidly converted in synaptosomes to phos-phorylatcd intermediates and was more slowly oxidized to 14CO2  相似文献   

16.
Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V max of 50.1 nmol/g/min and a K m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na+/H+ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na+-K+-2Cl/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co 2 of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na+/H+ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl/HCO3 exchange, lead to net NaCl secretion and obligate water transport into the brain.  相似文献   

17.
Abstract The addition of 1 mM glycine betaine to the growth medium of Chromatium sp. NCIMB 8379 relieved growth inhibition caused by exposure to supra-optimal Nad concentrations. Intracellular glycine betaine concentrations were dependent upon the NaCl concentration of the growth medium up to 3 M exogenous Nad. Kinetic data for the accumulation of [methyl-14C]-glycine betaine demonstrated that Chromatium sp. NCIMB 8379 possesses a constitutively expressed active transport system for glycine betaine. The transport system was saturable with respect to glycine betaine concentration and exhibited typical Michaelis-Menten type kinetics: K m= 24 μ M, V max= 306 nmol min−1 mg protein−1 at an external NaCl concentration of 1 M. The rate of glycine betaine transport decreased progressively with increasing growth medium NaCl concentration. This transport system may represent an adaptive response to growth in high osmolarity environments in this halotolerant isolate, allowing accumulation of glycine betaine from the external cell environment or recycling synthesised glycine betaine which has passively diffused from the cell.  相似文献   

18.
Transport of Histidine into Synaptosomes of the Rat Central Nervous System   总被引:4,自引:4,他引:0  
Abstract: Histidine transport into synaptosomes was studied in order to characterize this aspect of histamine synthesis in neurons. Histidine transport was found to be independent of sodium, calcium, and magnesium ions and dependent upon potassium and chloride ions. Histidine transport was also found to be energy dependent, and subcellular fractionation studies suggested it was highly localized to nerve terminals. Kinetic analysis of histidine transport in several brain regions indicated the presence of two uptake sites, a high-affinity site with a K m of approximately 35 μ M and a low-affinity site with a K m in the millimolar range. Density of the high-affinity site, as reflected by Vmax, correlates well with density of proposed histaminergic innervation. Rate of histidine transport was not altered by prior depolarization of the synaptosomes, indicating that histidine transport probably does not play a regulatory role in histamine synthesis.  相似文献   

19.
20.
Abstract: Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy- and temperature-dependent and saturable with a K m of 18.83 ± 3.31 m M and a V max of 4.78 ± 0.67 nmol/mg of protein/min. Treatment with ouabain (Na+,K+-ATPase inhibitor) or removal of extracellular Na+ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion-channel agents on agmatine uptake, only Ca2+-channel blockers inhibited the uptake, whereas a reduction in extracellular Ca2+ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na+-independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.  相似文献   

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