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1.
Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH exchange (or phenomenologically indistiguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pHint/pHext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+ K m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H+ (OH) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F and F–2 is electroneutral. Kinetic parameters for F and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pHext 5.8 (K m =1.33vs. 1.70 m;V max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV max, but does not affect thatK m, for carrier-mediated F transport. These data are consistent with similarK i's for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H+ K m (or OH IC50) corresponding to pH 4.89. External pH affects theV max, but not theK m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH or H+) rather than competitive binding that is mutually exclusive.  相似文献   

2.
Summary In jejunal brush-border membrane vesicles, an outwardly directed OH gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH exchange (or phenomenologically indistinguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pHint/pHext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+ K m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H+ (OH) at low pH (monovalent F predominates) and at least 2 H+ (OH) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F and F–2 is electroneutral. Kinetic parameters for F and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pHext5.8 (K m =1.33vs. 1.70 M;V max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV max, but does not affect theK m for carrier-mediated F transport. These data are consistent with similarK i ' s for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H+ K m (or OH lC50) corresponding to pH 4.89. External pH effects theV max, but not theK m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH or H+),rather than competitive binding that is mutually exclusive.  相似文献   

3.
Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [3H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na+ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K m =0.12, 0.11 and 0.10 m andJ max=0.01, 0.02 and 0.03 pmol·l–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K m =0.16 and 0.51 m andJ max=0.01 and 0.04 pmol·l–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.  相似文献   

4.
We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca2+ uptake was inhibited by TG up to 10 nm (apparent Ki4.2 nm, Ca2+ pool I). An additional increase of inhibition up to 85–90% of total Ca2+ uptake could be achieved at 15 to 20 nm of TG (apparent Ki12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30–40% of total Ca2+ uptake was inhibited by 100 m of vanadate (apparent Ki18 m, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki300 m) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at 25% over a wide range of Ca2+ filling. About 10–20% remained unreleasable by IP3. Reduction of IP3 releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki 3.0 nm for IP3-induced Ca2+ release as compared to 4.2 nm for Ca2+ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations >10 nm which blocked Ca2+ pool II the apparent Ki values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent Ki values were 18 m for Ca2+ uptake and 7 m for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were 300 and 200 m, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 m. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 m. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 m. These data allow the conclusion that there are at least three different Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and IP3 insensitive but highly vanadate-sensitive Ca2+ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca2+ pools with different TG-, vanadate- and IP3-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.  相似文献   

5.
Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10-2 mol l–1, gibberellic acid (GA3) 3.10-2 mol l–1, and 6-benzylaminopurine (BA) 2 mol l–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l–1, GA3 14 mol l–1, and kinetin 5 mol l–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l–1 and BA 10 mol l–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy  相似文献   

6.
Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. 36Cl uptake into these vesicles was studied in the presence of an outwardly directed Cl gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC50 values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced 36Cl uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC50 of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced 36Cl uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl uptake with IC50 values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl gradient and this channel is sensitive to Cl channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.  相似文献   

7.
Summary To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ·cm2, and short circuit current (I sc=2–20 A/cm2), representing active secretion of Cl, increased >threefold with addition of 10 m isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescencevs. [Cl] (0–90mm) was carried out using high-K buffers containing the ionophores nigericin (5 m) and tributyltin (10 m); SPQ fluorescence was quenched with a Stern-Volmer constant of 13m –1. Intracellular Cl activity was 43±4mm. Cl flux was measured in response to addition and removal of 114mm Cl from the bathing solution. Addition of 10 m isoproterenol increased Cl efflux from 0.10 to 0.27mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1mm). In the absence of isoproterenol, removal of external Na or addition of 0.5mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.  相似文献   

8.
The presence of a folate binding protein of high-affinity type (affinity constant 5 · 109M–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand3H-folate. Dissociation of3H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M r 25 000) and a minor one (M r 100 000).  相似文献   

9.
Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK 1=0.67±0.16 m,k 2=1.6±0.7 sec–1,k –2=0.17±0.09 sec–1,K 1=6.3±1.7 m,k 2=9±4 sec–1 andk –2=7±3 sec–1. The apparent dissociation constants of chloride from band 3,K Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.  相似文献   

10.
Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3±0.1 m) for H2-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of DBDS is virtually the same as the ID50 (0.47±0.04 m) for H2-DIDS inhibition of red cell Cl flux, thus relating DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID50 for cytochalasin B modulation of DBDS is 0.1±0.2 m in good agreement withK D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.  相似文献   

11.
Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K+-stimulated, Ca2+-dependent release of [3H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [3H]dopamine. Local anesthetics were shown to inhibit uptake of [3H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine  相似文献   

12.
The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe2+ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg2+, 20 m Ca2+ and 3.6 m Mn2+. Chromate reduction was not affected by the addition of MgCl2, CdCl2, ZnCl2, MnCl2, Na2SO4 (1000 m), and Na2MoO4 (100 m) to the activity assays. However, activity was inhibited by the presence of Na2SO4 (10 mm), Na2MoO4 (200 m) and ferric citrate.  相似文献   

13.
Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K m = 3 m and low capacity (Vmax) 0.4 mol l-Glu · Kg–1 dry cell mass · 10 min–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X AG family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K m 39 m but high capacity (V max) 1.8 mol l-Glu · Kg–1 dry cell mass · 10 min–1.  相似文献   

14.
A method of measuring CO2gas exchange (caused, for example, by microalgal photosynthesis on emersed tidal mudflats) using open flow IR gas analyzers is described. The analyzers are integrated in a conventional portable photosynthesis system (LI-6400, LI-COR, Nebraska, USA), which allows manipulation and automatic recording of environmental parameters at the field site. Special bottomless measuring chambers are placed directly on the surface sediment. Measurements are performed under natural light conditions and ambient CO2concentrations, as well as under different CO2concentrations in air, and various PAR radiation levels produced by a LED light source built into one of the measurement chambers. First results from tidal channel banks in a north Brazilian mangrove system at Bragança (Pará, Brazil) under controlled conditions show a marked response of CO2assimilation to CO2concentration and to irradiance. Photosynthesis at 100molmol–1CO2in air in one sample of a well-developed algal mat was saturated at 309mol photons m–2s–1, but increased with increasing ambient CO2concentrations (350 and 1000mol mol–1CO2) in the measuring chamber. Net CO2assimilation was 0.8mol CO2m–2s–1at 100mol mol–1CO2, 5.9mol CO2m–2s–1at 350mol mol–1CO2and 9.8mol CO2m–2s–1at 1000mol mol–1CO2. Compensation irradiance decreased and apparent photon yield increased with ambient CO2concentration. Measurements under natural conditions resulted in a quick response of CO2exchange rates when light conditions changed. We recommend the measuring system for rapid estimations of benthic primary production and as a valuable field research tool in connection with certain ecophysiological aspects under changing environmental conditions.  相似文献   

15.
Summary We have used a combination of chemical labeling and detergent fractionation techniques to locate the divalent cation binding sites on the chloroplast membrane. We determined the Ca2+-binding properties of Triton X-100 subchloroplast particles. Photosystem II (TSFII) particles showed one binding site withn=8.4 moles-mg chl–1 andk d =20 m. Photosystem I (TSFI) particles contained two binding sites. The first had ann=1.5 moles-mg chl–1 andk d =4 m. The second had ann=9.6 moles-mg chl–1 andk d =160 m. We have previously shown (Prochaska & Gross,Biochim. Biophys. Acta 376:126, 1975) that the divalent cation binding sites could be blocked using a water-soluble carbodiimide plus a nucleophile. Chlorophylla fluorescence and lightscattering changes were affected at the same carbodiimide concentrations emphasizing the relationship between these processes. The carbodiimide-sensitive sites were found to be located on the Photosystem II particles. A direct correlation between the inhibition of calcium binding and the carbodiimide-mediated incorporation of a (14C)-nucleophile was observed upon varying such parameters as carbodiimide concentration, nucleophile concentration, pH, and time of reaction. The presence of CaCl2 during the carbodiimide plus nucleophile modification procedure decreased the incorporation of (14C)-nucleophile, emphasizing the competition of the CaCl2 and the modification reagents for some of the same sites. Sodium dodecylsulfate gel electrophoresis of chlorophyll protein aggregates suggested that the site of competition of the calcium chloride and the modification reagents was the light-harvesting chlorophylla/b protein.  相似文献   

16.
High-affinity folate binding in human prostate   总被引:1,自引:0,他引:1  
Binding of3H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (Mr100 and 25kDa), but only one single band (Mr65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum3H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n=6).  相似文献   

17.
Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [3H]-histamine uptake yielded a Km of 0.19±0.03 M and a Vmax of 3.12±0.75 pmol×mg protein–1×min–1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na+ with choline inhibited the initial rate of uptake showing a strong Na+-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K+-gradient (in>out) by high K+ or by K+-channel blockers, BaCl2, (100 M), quinine (100 M) or Sparteine (20 M) produced also remarkable inhibitions in the uptake of [3H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 M with a Ki value of 2.8 M, indicating the specificity of the uptake. [3H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33±0.08 M; and a Vmax of 2.65±0.35 pmoles×mg protein–1×min–1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [3H]histamine efflux was sensitive to Na+ omission, ouabain treatment and high K+ or K+ channel blockers, resulting in marked elevations in the efflux. Data indicate that glial uptake of histamine is a high affinity, Na+-dependent and electrogenic, driven by an inward-oriented sodium ion gradient and an outward-oriented potassium ion gradient and functions as part of histamine inactivation, at least in a shunt mechanism.Abbreviations used HA histamine - [3H]HA [2.5-3H]-histamine - dl--aAA dl-alpha-aminoadipic acid - (Na++K+) ATP-ase sodium and potassium activated adenosine triphosphatase - SAH S-Adenosyl-d-Homocysteine - HNMT histamine-N-methyltransferase  相似文献   

18.
Summary Chlamydomonas reinhardtii cells provide an effective system for glycolate photoproduction, operative during 30 h when they are growing under low CO2, in the presence of 1 mM aminooxyacetate and 50 M acetazolamide. Glycolate excretion by the cells can proceed for about 4 days if every other 12 h a high CO2 level is restored in the culture in the absence of inhibitors. The immobilized system in alginate beads has about a twofold higher glycolate photoproduction rate (23 mol·mg chlorophyll (Chl)–1·h–1) than free-living cells (12 mol · mg Chl–1 · h–1). Offprint requests to: C. Vílchez  相似文献   

19.
Summary We have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K 0.510 or 30 m, depending on the method for [Ca2+] i determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK 0.52.5 m. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca2+] i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca2+ channel blocker La3+ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP3 formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.  相似文献   

20.
Summary Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20°C over a limited range of CAR concentrations were characterized by aK m of 350 m and a maximal velocity (V) of 780 m·min–1 (V/K m =2.28 min–1). Equilibrium exchange at 20°C over a wider range of concentrations was best described by a saturable component with aK m of 500 m and av of 1230 m·min–1 (V/K m =2.26 min–1) and either a saturable component of highK m or a nonsaturable component ofk=0.3 min–1. For the saturable component, thev/K m values were similar in both procedures.CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (K i <0.5nm) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurialp-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. These effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.  相似文献   

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