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1.
Jittima Weerachayaphorn 《生物化学与生物物理学报:生物膜》2008,1778(4):1051-1059
Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1. 相似文献
2.
Experiments on the effects of varying concentrations of Ca2+ on the Mg2+ + Na+-dependent ATPase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase showed that Ca2+ was a partial inhibitor of this activity. When Ca2+ was added to the reaction mixture instead of Mg2+, there was a ouabain-sensitive Ca2+ + Na+-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg2+ + Na+-dependent activity. The apparent affinities of the enzyme for Ca2+ and CaATP seemed to be higher than those for Mg2+ and MgATP. Addition of K+, along with Ca2+ and Na+, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca2+ + Na+ + K+-dependent ATPase was about two orders of magnitude smaller than that of Mg2+ + Na+ + K+-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca2+, Na+, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K+. The enzyme also exhibited a Ca2+ + Na+-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca2+ on Mg2+-dependent activities, nor the Ca2+-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg2+ in all reaction mixtures, it could not be determined whether the apparent Ca2+-dependent activities involved enzyme-substrate complexes containing Ca2+ as the divalent cation or both Ca2+ and Mg2+. 相似文献
3.
Triton X-100 extract from brush border membranes of rat small intestine was recombined with egg phosphatidylcholine liposomes by the freeze-thaw sonication method. The treated liposomes showed a Na+-dependent uptake of cyclacillin, which was inhibited by a low concentration of mercuric ions and L-phenylalanylglycine, but not by glycine. These are consistent with the absorption characteristics of the antibiotic in situ and indicate that reconstitution of the Na+-dependent active cyclacillin transport system of rat small intestine has been achieved. 相似文献
4.
Our previous studies showed that when ethylmercurithiosalicylate (thimerosal) interacts with the transport ATPase of the guinea pig kidney under specified conditions, the Na+ + K+-dependent ATPase activity is inhibited, while the Na+-dependent ATPase, the Na+ + ATP-dependent phosphorylation of the enzyme, and the K+-dependent discharge of the phosphoenzyme seem to be unaffected. Here we describe other properties of the thimerosal-treated enzyme: Na+-dependent ADP-ATP exchange, Na+-dependent UTPase, and K+-dependent p-nitrophenylphosphatase activities of the modified enzyme are not inhibited. Kinetics of the Na+ effect on the UTPase activities of the native and the modified enzyme are the same. However, K+ has a greater inhibitory effect on the Na+-UTPase of the modified enzyme than on the Na+-UTPase of the native enzyme. The increase in the apparent affinity of the thimerosal-treated enzyme for K+ is also evident from the kinetics of the K+ effect on p-nitrophenylphosphatase. Neither the native enzyme nor the modified enzyme catalyzes a P1-ATP exchange. The uninhibited activities of the thimerosal-treated enzyme are sensitive to ouabain. These data provide further support for those reaction mechanisms in which the existence of two ATP sites within the enzyme is assumed. 相似文献
5.
Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na+-dependent inorganic phosphate (Pi) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na+-dependent 32Pi cotransport in cultured fetal rat cortical neurons, where >90% of saturable Pi uptake is Na+-dependent. Saturable, Na+-dependent 32Pi uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na+-dependent Pi transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent Km values for Pi and Na+ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca2+ markedly reduced (>60%) Na+-dependent Pi uptake, with a threshold for maximal Pi import of 1–2.5 mM CaCl2. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na+ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na+. Furthermore, a substantial fraction of the 32Pi cotransported with Na+ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na+-dependent Pi import rates. These data establish the presence of a Na+-dependent Pi cotransport system in neurons of the CNS, demonstrate the Ca2+-dependent nature of 32Pi uptake, and suggest that the neuronal Na+-dependent Pi cotransporter may import Pi required for the production of high-energy compounds vital to neuronal metabolism. 相似文献
6.
Summary Treatment of red cell membranes with pure phospholipase C inactivates (Na++K+)-ATPase activity and Na+-dependent phosphorylation but increases K+-dependent phosphatase activity. When phospholipase A2 replaces phospholipase C, all activities are lost. Activation of K+-dependent phosphatase by treatment with phospholipase C is caused by an increase in the maximum rate of hydrolysis ofp-nitrophenylphosphate and in the maximum activating effect of K+, the apparent affinities for substrate and cofactors being little affected. After phospholipase C treatment K+-dependent phosphatase is no longer sensitive to ouabain but becomes more sensitive to N-ethylmaleimide. In treated membranes Na+ partially replaces K+ as an activator of the phosphatase. Although ATP still inhibits phosphatase activity, neither ATP nor ATP+Na+ are able to modify the apparent affinity for K+ of K+-dependent phosphatase in these membranes. 相似文献
7.
Joseph T. Tupper 《生物化学与生物物理学报:生物膜》1975,394(4):586-596
In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30% of the total fux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22% of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of α-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A. 相似文献
8.
R.M. Johnstone 《生物化学与生物物理学报:生物膜》1975,413(2):252-264
Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence of gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+. 相似文献
9.
R. S. Prather M. S. Peters L. J. Van Winkle 《Molecular reproduction and development》1993,34(3):250-254
Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na+-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system BO,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na+-dependent in the oocyte; this Na+-dependent component of transport becomes undetectable by the blastocyst stage. The Na+-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system BO,+. In both oocytes and blastocysts Na+-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na+-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na+-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na+-dependent system BO,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development. © 1993 Wiley-Liss, Inc. 相似文献
10.
The Na+-dependent transport of 5-oxoproline into rabbit renal brush-border vesicles was stimulated by a K+ diffusion potential (interior-negative) induced by valinomycin. Na+ salts of two anions of different epithelial permeabilities also affected 5-oxoproline transport. These results show that the Na+-dependent 5-oxoproline transport in renal brush-border vesicles is an electrogenic process which results in a net transfer of positive charge. Maximum transport of 5-oxoproline occurred at an extravesicular pH of 6.0 to 8.0 and over that pH range, 5-oxoproline exists completely as an anion with a negative charge. The simplest stoichiometry consistent with this process is, therefore, the cotransport of one 5-oxoproline anion with two sodium ions. The presence of K+ inside the vesicles stimulated the Na+-dependent transport of 5-oxoproline. This stimulatory effect was specific for K+ and required the presence of Na+. The presence of Na+ gradient was not mandatory for the K+ action. The stimulation by the intravesicular K+ was seen in the presence as well as in the absence of a K+ gradient. Therefore, the increased influx of 5-oxoproline was not coupled to the simultaneous efflux of K+. The presence of K+ in the extravesicular medium alone did not affect the Na+-dependent transport of 5-oxoproline, showing that the site of K+ action was intravesicular. Glutamate did not interact with the Na+-dependent 5-oxoproline transport even in the presence of an outward K+ gradient. 相似文献
11.
Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris 总被引:33,自引:13,他引:20
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The pH-dependent fluorescence quenching of acridine orange was used to study the Na+- and K+-dependent H+ fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na+-dependent H+ flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na+ concentration and was competitively inhibited by amiloride (Ki = 0.1 millimolar). The potential-dependent component of H+ flux showed an approximately linear dependence on Na+ concentration. In contrast, the K+-dependent H+ flux apparently consisted of a single component which showed an approximately linear dependence on K+ concentration, and was insensitive to amiloride. Based on the Na+- and K+-dependent H+ fluxes, the passive permeability of the vesicle preparation to Na+ was about half of that to K+.
The apparent Km for Na+ of the electroneutral Na+/H+ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na+/H+ antiport which can account for the estimated in vivo accumulation ratio for Na+ into the vacuole.
相似文献12.
Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis 总被引:10,自引:10,他引:0
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At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na+. The rate of photosynthesis greatly exceeded the CO2 supply rate and indicated that HCO3− was taken up by a Na+-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na+ and exceeded the CO2 supply rate by 8 to 45 times. The apparent photosynthetic affinity (K½) for DIC was high (6-40 micromolar) and was not markedly affected by Na+ concentration, whereas with air-grown cells K½ (DIC) decreased by more than an order of magnitude in the presence of Na+. Lithium, which inhibited Na+-dependent HCO3− uptake in air-grown cells, had little effect on Na+-independent HCO3− uptake by standing culture cells. A component of total HCO3− uptake in standing culture cells was also Na+-dependent with a K½ (Na+) of 4.8 millimolar and was inhibited by lithium. Analysis of 14C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na+-independent CO2 transport system. The conversion from Na+-independent to Na+-dependent HCO3− uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO3− for subsequent photosynthetic fixation. 相似文献
13.
Resting cells ofFusobacterium nucleatum ATCC 10953, when provided with glutamic acid (Na+ salt) as fermentable energy source, rapidly accumulated [14C]glucose, from the medium. Sugar accumulation was not observed when Na+ glutamate was replaced by ammonium glutamate. However, addition of Na+ (chloride) to the latter system elicited uptake of [14C]glucose by the organism. Of other monovalent cations tested, only Li+ was found to be slightly stimulatory, but K+, Rb+, and Cs+ ions were ineffective. For determination of the role(s) of Na+ in sugar accumulation, the transport of [14C]glucose and [14C]glutamic acid by the cells was studied independently, with lysine as an alternate (and Na+-independent) energy source. In the presence of lysine, cells ofF. nucleatum 10953 accumulated [14C]glucose from a Na+-free medium, but, in contrast, uptake and fermentation of [14C]glutamic acid was Na+-dependent. The glucose transport system is Na+-independent. However, our data indicate dual role(s) for Na+ in the transport and intracellular metabolism of glutamic acid. The Na+-dependent glutamate fermentation pathway provides the necessary energy for active transport of glucose by the resting cell. 相似文献
14.
Transport of selenate and selenite across the brush border membrane of rat and sheep small intestine
F. Ardüser S. Wolffram E. Scharrer B. Schneider 《Biological trace element research》1986,9(4):281-290
Mucosal uptake of75Se-labeled selenate and selenite across the brush border was investigated in sheep and rat small intestine, using 3-min mucosal exposures. Uptake of selenate and selenite occurred faster in rat than in sheep small intestine. With the exception of sheep duodenum, mucosal selenate uptake was Na+-dependent in sheep and rat small intestine. Mucosal uptake of selenite across the brush border was Na+-dependent only in sheep midjejunum, whereas it was Na+-independent in sheep duodenum and ileum and the rat whole small intestine. Various anions inhibited selenate transport in the presence of Na+ in sheep midjejunum in the order S2O2 2- = CrO4 2- > MoO4 2- and in rat ileum in the order CrO4 2- = S2O3 2- > SC4 2- > MoO4 2-. Thiosulfate also inhibited mucosal selenite uptake in the presence of Na+ in sheep midjejunum. Preincubation of rat ileum with glutathione (GSH) enhanced mucosal selenite uptake, whereas selenate uptake remained unaffected. These results indicate that selenate transport across the brush border membrane is energized in part by the Na+-gradient. Moreover, the Na+-dependent transport mechanism for the Se salts apparently has an affinity for other anions (S2O3 2-, SO4 2-, CrO4 2-, MOo4 2-). The findings further indicate that intracellular GSH plays a role in the absorption of selenite, probably by an increase of intracellular selenite metabolism. The Na+-independent mucosal uptake of selenate and selenite probably represents diffusion. 相似文献
15.
Intracellular Ca++ is known to influence Na+ flux in luminal membranes. Abnormally elevated Ca++ levels in some cells is believed to be the primary pathophysiologic defect in cystic fibrosis (CF). This in turn is thought to alter Na+ transport which accounts for certain clinical manifestations of this disease. Two Na+-dependent intestinal transport mechanisms have been reported to be suppressed or missing in CF. To examine whether alterations in cell Ca++ may account for these findings, studies were performed to examine the influence of Ca++ on Na+-solute co-transport across intestinal luminal membranes. Purified brush border membrane vesicles prepared from rat small bowel were preincubated in either Ca++-free buffer or buffer containing 2.5 mM CaCl2. Ca++ loaded vesicles showed marked inhibition of Na+ co-transport of taurocholic acid, taurochenodeoxycholic acid, glucose and valine when compared to controls. The uptake of Na+ was also significantly reduced by intravesicular Ca++. These data demonstrate that intravesicular Ca++ inhibits Na+-coupled solute transport as well as Na+ influx across intestinal brush border membranes. These data suggest that intracellular Ca++ may suppress Na+-dependent solute absorption in the intestine. Results presented here further support the theory that elevated intracellular Ca++ may account for intestinal malabsorption and other altered transport phenomena reported in CF. 相似文献
16.
The specific activity of (Na+ + Mg2+)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na+ + K+ + Mg2+)-dependent ATPase in both cases.(Na+ + Mg2+)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI2, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na+ + Mg2+)-dependent ATPase activity of gills arises from the presence of a (Na+ + K+ + Mg2+)-dependent ATPase. 相似文献
17.
The uptake of l-glutamic acid into brush-border membrane vesicles isolated from rat renal proximal tubules is Na+-dependent. In contrast to Na+-dependent uptake of d-glucose, pre-equilibration of the vesicles with K+ stimulates l-glutamic acid uptake. Imposition of a K+ gradient () further enhances Na+-dependent l-glutamic acid uptake, but leaves K+-dependent glucose transport unchanged. If K+ is present only at the outside of the vesicles, transport is inhibited. Intravesicular Rb+ and, to a lesser extent, Cs+ can replace intravesicular K+ to stimulate l-glutamic acid uptake. Changes in membrane potential incurred by the imposition of an H+-diffusion potential or anion replacement markedly affect Na+-dependent glutamic acid uptake only in the presence of K+. Experiments with a potential-sensitive cyanine dye also indicate that, in the presence of intravesicular K+ a charge movement is involved in Na+-dependent transport of l-glutamic acid.The data indicate that Na+-dependent l-glutamic acid transport can be additionally energized by a K+ gradient. Furthermore, intravesicular K+ renders Na+-dependent l-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference. 相似文献
18.
Data are presented which prove that 3-O-methylfluorescein phosphate is a substrate for the K+-dependent phosphatase that is associated with Na+,K+-ATPase. Conditions for the continuous fluorimetric assay of 3-O-methylfluorescein phosphatase are described. Enzyme preparations from three different tissues with widely different specific activities exhibit similar Km values for 3-O-methylfluorescein phosphate. Correlation between Na+,K+-ATPase activity and K+-dependent 3-O-methylfluorescein phosphatase activity is demonstrated in several partially purified enzyme preparations and crude tissue fractions. When the K+-dependent 3-O-methylfluorescein phosphatase of a crude rat-brain homogenate is assayed, the activity is a linear function of the amount of homogenate added to the assay mixture. The equivalent of 10 μg of brain tissue may be assayed under the conditions used. The potential value of this highly sensitive fluorimetric method for the assay of enzyme in small samples of various tissues is suggested. 相似文献
19.
PbCl2 in micromolar concentrations stimulates phosphorylation of electroplax microsomal protein in the absence of Na+. Other divalent cations showed little or no such effect. The (Mg2+ + Pb2+)- and (Mg2+ + Na+)-dependent membrane-bound protein kinase activities in electroplax particulate preparations exhibit properties in common, including their acid stability, ouabain sensitivity, ATP specificity, and molecular size. It is concluded that the (Mg2+ + Pb2+)-dependent phosphoprotein is part of the Na+-, K+-dependent adenosine triphosphatase [(NaK)ATPase]. The Pb2+-dependent product, in contrast to the Na+-dependent one, is insensitive to K+ and the hydrolysis of ATP is thus inhibited. 相似文献
20.
Marco Antonio Lacerda-Abreu Thais Russo-Abrahão José Roberto Meyer-Fernandes 《Cell biology international》2021,45(8):1768-1775
Metastasis is a major cause of death in patients with breast cancer. A growing body of evidence has demonstrated the antitumour effects of resveratrol, a non-flavonoid polyphenol. Resveratrol inhibits metastatic processes, such as the migration and invasion of cancer cells. In several cancer types, the importance of inorganic phosphate (Pi) for tumor progression has been demonstrated. The metastatic process in breast cancer is associated with Na+-dependent Pi transporters. In this study, we demonstrate, for the first time, that resveratrol inhibits the Na+-dependent Pi transporter. Results from kinetic analysis shows that resveratrol inhibits Na+-dependent Pi transport non-competitively. Resveratrol also inhibits adhesion/migration in MDA-MB-231 cells, likely related to inhibition of the Na+-dependent Pi transporter. 相似文献