首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.  相似文献   

2.
Zinc transport into brush-border membrane vesicles was investigated by measuring uptake rates at a very short incubation time (2 seconds), during the initial linear uptake. A divalent cation chelator (EGTA) was added to the stop and washout solutions in order to remove the zinc bound to the external surface of the vesicles. Under these conditions, we showed that zinc enters the vesicles by (1) a saturable carrier-mediated process, and (2) an unsaturable pathway. The kinetic parameters we calculated were an affinity of 0.215 +/- 0.039 mM, a Jmax of 17.2 +/- 1.7 nmol.min-1.(mg protein)-1 and an unsaturable constant of 0.025 +/- 0.006 (n = 6). The imposition of an outwardly directed K+ gradient (negative inside) did not affect the Jmax value of the zinc uptake but increased the Km value significantly. This suggests that, at least a portion of zinc which crosses the membrane does not do so in a cationic form. Zinc uptake was decreased or increased according to the nature of accompanying anions (Cl-, SO4(2)-, SCN-) in the absence of any membrane potential. With highly permeant anions such as thiocyanates, zinc uptake was considerably augmented, suggesting a movement of zinc in a complexed form involving the presence of negative species. We also showed that cadmium competitively inhibited the zinc uptake; we measured a Ki value of 0.21 mM, indicating a similar affinity of cadmium for the carrier as zinc itself. By contrast, the presence of calcium had little effect on zinc entry into vesicles. The calcium ionophore A23187 had only a slight stimulating effect on zinc uptake. These results indicate that zinc and calcium transports are probably independent of each other.  相似文献   

3.
Integumentary uptake of [3H]-L-histidine by Nereis succinea was measured in the presence and absence of selected heavy metals and the amino acid L-leucine in 60% artificial seawater (ASW). The time course of 10 microM [3H]-L-histidine uptake into worms over a 60 min incubation was approximately doubled in the presence of 0.5 microM zinc and when calcium in the incubation medium was reduced from 6 mM to 5 microM the stimulatory effect of zinc on amino acid accumulation was reduced and uptake under the latter conditions was approximately half that of the control. Zinc stimulation of [3H]-L-histidine influx was a hyperbolic function of zinc concentration over the range 0 to 50 microM metal and displayed an apparent activation or affinity constant of 385+/-127 nM Zn(2+). The hyperbolic stimulatory effect of 1 microM Zn(2+) on the time course of 10 microM [3H]-L-histidine uptake was abolished in the presence of 25 microM L-leucine, suggesting that this amino acid shared the same transport system as [3H]-L-histidine and acted as a potential competitive inhibitor. Influx of [3H]-L-histidine was a hyperbolic function of external amino acid concentration and displayed an apparent affinity constant (Km) of 23.71+/-5.02 microM and an apparent aximal velocity (J(max)) of 4701+/-449 pmol/g dry wt.x15 min. Addition of 0.5 microM zinc resulted in a four-fold increase in J(max) and a doubling of K(m), suggesting the effect of the metal was mostly on the rate of amino acid transport. [3H]-L-histidine influx was mildly stimulated by Fe(2+) (0.5 microM), but was unaffected by either Ag(+) or Al(3+) (both at 0.5 microM). These results suggest that [3H]-L-histidine uptake into worm integument may take place by the classical Na(+)-independent L-transport system shared by L-leucine and regulated by exogenous calcium and other divalent metal concentrations.  相似文献   

4.
Lobster (Homarus americanus) hepatopancreas is a complex, heterogeneous tissue composed of four epithelial cell types that individually contribute to the overall functional properties of digestion, absorption, secretion, and detoxification. Previous studies, using purified hepatopancreatic brush border membrane vesicles, have described the properties of an electrogenic, 2Na+/1H+ antiporter in this tissue that regulates the absorption and secretion of these cations. These studies were not able to localize this cation exchange phenomenon to specific epithelial cell types. In the present study, sodium/proton exchange by purified, single cell, suspensions of lobster (Homarus americanus) hepatopancreatic epithelium was investigated using a centrifugal elutriation method to cleanly separate the four individual cell types for subsequent physiological characterization. Results indicate that all four hepatopancreatic epithelial cell types possessed the 2Na+/1H+ antiporter as a result of its unique sigmoidal influx properties. Hill Coefficients, measures of transport sigmodicity obtained from kinetic analyses of 22Na+ influx by single cell type suspensions, varied from 1.56 +/- 0.30 (R-cell suspensions) to 2.79 +/- 0.41 (F-cell suspensions), suggesting that different numbers of sodium ions may be accommodated by each cell type. Both calcium and zinc were competitive inhibitors of 22Na+ influx in E-cells (calcium Ki = 105.1+/-5.2 microM; zinc Ki = 46.2 +/- 7.8 microM), but the extent to which these divalent cations inhibited monovalent cation transport by each cell type varied. It is concluded that different isoforms of the electrogenic 2Na+/1H+ antiporter may be present in each hepatopancreatic cell type and thereby contribute in differing degrees to the cation regulatory functions performed by the overall organ.  相似文献   

5.
ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.  相似文献   

6.
Calcium (Ca(2+)) transport by the distal tubule (DT) luminal membrane is regulated by the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytosolic surface of the membrane could influence the Ca(2+) channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca(2+) uptake by these vesicles loaded with ATP or the carrier. The presence of 100 microM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca(2+) uptake from 0.57 +/- 0.02 to 0.71 +/- 0.02 pmol/microg per 10 sec (P < 0. 01) in the absence of Na(+) and from 0.36 +/- 0.03 to 0.59 +/- 0.01 pmol/microg per 10 sec (P < 0.01) in the presence of 100 mM Na(+). This effect was dose dependent with an EC(50) value of approximately 40 microM. ATP action involved the high-affinity component of Ca(2+) transport, decreasing the Km from 0.08 +/- 0.01 to 0.04 +/- 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPgammas abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, we also investigated the effect of intravesicular cytochalasin on Ca(2+) transport. Inclusion of 20 microM cytochalasin B decreased 0.5 mM Ca(2+) uptake from 0.33 +/- 0.01 to 0.15 +/- 0.01 pmol/microg per 10 sec (P< 0.01). However, when both 100 microM ATP and 20 microM cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca(2+) uptake by the PT luminal membrane. We conclude that the high-affinity Ca(2+) channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca(2+) channels within this membrane.  相似文献   

7.
Na+, Li+ and Cl− transport by brush border membranes from rabbit jejunum   总被引:1,自引:0,他引:1  
Na+, Li+, K+, Rb+, Br-, Cl- and SO4(2-) transport were studied in brush border membrane vesicles isolated from rabbit jejunum. Li+ uptakes were measured by flameless atomic absorption spectroscopy, and all others were measured using isotopic flux and liquid scintillation counting. All uptakes were performed with a rapid filtration procedure. A method is presented for separating various components of ion uptake: 1) passive diffusion, 2) mediated transport and 3) binding. It was concluded that a Na+/H+ exchange mechanism exists in the jejunal brush border. The exchanger was inhibited with 300 microM amiloride or harmaline. The kinetic parameters for sodium transport by this mechanism depend on the pH of the intravesicular solution. The application of a pH gradient (pHin = 5.5, pHout = 7.5) causes an increase in Jmax (50 to 125 pmol/mg protein . sec) with no change in Kt (congruent to 4.5 nM). Competition experiments show that other monovalent cations, e.g. Li+ and NH4+, share the Na+/H+ exchanger. This was confirmed with direct measurements of Li+ uptakes. Saturable uptake mechanisms were also observed for K+, Rb+ and SO4(2-), but not for Br-. The Jmax for K+ and Rb+ are similar to the Jmax for Na+, suggesting that they may share a transporter. The SO4(2-) system appears to be a Na+/SO4(2-) cotransport system. There does not appear to be either a Cl-/OH- transport mechanism of the type observed in ileum or a specific Na+/Cl- symporter.  相似文献   

8.
High affinity Ca2+ -Mg2+ ATPase in the distal tubule of the mouse kidney   总被引:1,自引:0,他引:1  
The purpose of this study was to investigate whether Ca2+ -Mg2+ ATPase in the distal tubule (where calcium transport is active, against a gradient, and hormone dependent) presents some characteristics different from those observed in the proximal tubule, and whether these characteristics are likely to shed light on the respective roles of this enzyme at the two sites of the nephron. The Ca2+ - and Mg2+-dependent ATP hydrolysis was measured in microdissected segments of the distal nephron, the kinetic parameters were determined, and the influence of magnesium upon the sensitivity to calcium was examined. Results were compared with those obtained in the proximal tubule, and in purified membranes as reported by others. In the distal tubule, low concentrations of Mg2+ (less than 10(-7) M) did not influence ATP hydrolysis. At concentrations above 10(-7) M, Mg2+ increased ATP hydrolysis according to Michaelis kinetics (apparent Km = 11.3 +/- 2.4 microM, Vmax = 219 +/- 26 pmol.mm-1.20 min-1). The addition of 1 microM Ca2+ decreased the apparent Km for Mg2+ and the Vmax for Mg2+. Similar results were obtained in the proximal tubule. At low Mg2+ concentrations, Ca2+ also stimulated ATP hydrolysis according to Michaelis kinetics with an apparent Km value for Ca2+ of 0.18 +/- 0.06 and 0.10 +/- 0.03 microM Ca2+ (ns) and a Vmax of 101 +/- 12 and 89 +/- 9 pmol.mm-1.20 min-1 (ns) in the distal and proximal tubules, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
Calcium influx in internally dialyzed squid giant axons   总被引:9,自引:4,他引:5       下载免费PDF全文
A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant [Ca++]i. At a free [EGTA]i greater than 200 microM, the measured resting Ca influx and the expected increment in Ca entry during electrical stimulation were independent of the axoplasmic free [EGTA]. To avoid Ca uptake by the mitochondrial system, cyanide, oligomycin, and FCCP were included in the perfusate. Axons dialyzed with a standard medium containing: [ATP] = 2 mM, [Ca++]i = 0.06 microM, [Ca++]o = 10 mM, [Na+]i = 70 mM, and [Na+]o = 465 mM, gave a mean Ca influx of 0.14 +/- 0.012 pmol.cm-2.s-1 (n = 12. Removal of ATP drops the Ca influx to 0.085 +/- 0.007 pmol.cm-2.s-1 (n = 12). Ca influx increased to 0.35 pmol.cm-2,s-1 when Nao was removed. The increment was completely abolished by removing Nai+ and (or) ATP from the dialysis medium. At nominal zero [Ca++]i, no Nai-dependent Ca influx was observed. In the presence of ATP and Nai [Ca++]i activates the Ca influx along a sigmoid curve without saturation up to 1 microM [Ca++]i. Removal of Nai+ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]i (0.087 +/- 0.008 pmol.cm-2.s-1; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Nai+, Cai++, and ATP, sensitive to membrane potential, and partially inhibited by external Co++.  相似文献   

10.
The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.  相似文献   

11.
Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.  相似文献   

12.
Noll M  Lutsenko S 《IUBMB life》2000,49(4):297-302
All cells have developed various mechanisms to regulate precisely the availability of important micronutrients such as zinc and copper; in many cells, this regulation is mediated by P1-type ATPases. Most of the P1-ATPases have been described very recently, and little is known about their molecular mechanism and regulation. Here, we demonstrate that the expression of ZntA, a Zn,Cd-transporting P-type ATPase of Escherichia coli, is specifically regulated by the transported cations, cadmium and zinc. Nickel, cobalt, and copper did not induce the expression of ZntA, even when present at concentrations as high as 0.6-1 mM. The effect of zinc and cadmium on the ZntA expression is concentration dependent, the apparent Km for Cd (19 microM) being markedly lower than that for Zn (100 microM). This metal selectivity is opposite to the known metal selectivity of transport by ZntA. Thus, we speculate that, to maintain zinc concentrations in the cell in the presence of cadmium, ZntA probably interacts with other proteins that modulate the ZntA selectivity towards transported cations.  相似文献   

13.
Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.  相似文献   

14.
Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 microM), the initial rate of 65Zn2+ accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.  相似文献   

15.
Extracellular zinc (Zn)-binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts. The uptake of alpha 2-macroglobulin, a major serum Zn-binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate. The fibroblast growth medium, BME with 10% FBS, contains several Zn-binding ligands. These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator. Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW less than 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration. Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the Ka value for the ligands and therefore proportional to the free Zn concentration. When histidine and desferrioxamine, two structurally different Zn-binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar Vmax and Km values (+/- S.E.M.) of 373 +/- 6 pmol/micrograms DNA/h and 0.08 +/- 0.004 microM for histidine, and 349 +/- 10 pmol/micrograms DNA/h and 0.06 +/- 0.008 microM for DFO, suggesting that the same transport mechanism was operating in both systems. We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that "free" Zn is acquired by the cell.  相似文献   

16.
Acyclovir transport into human erythrocytes   总被引:2,自引:0,他引:2  
The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.  相似文献   

17.
To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi.mmol-1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27-39 pmol.g-1.min-1 and 2.6-4.8 microM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol.g-1.min-1, thus representing a significant component of thiamine supply to nervous tissue.  相似文献   

18.
Zinc Uptake into Synaptosomes   总被引:4,自引:0,他引:4  
Zinc uptake was studied in synaptosomes, isolated by the Ficoll flotation technique, using the radiotracer 65Zn. True uptake of zinc could be discriminated from binding to the outside of the synaptosomes by the absence of accumulation at 0 degree C and the dependency of the rate of uptake on the medium osmolarity. The zinc uptake, studied in the presence of various zinc-complexing agents, showed saturation kinetics when analyzed in terms of [Zn]free, yielding Km = 0.25 microM. The zinc uptake was independent of both ATP and the Na+ gradient. No efflux of zinc could be demonstrated from preloaded synaptosomes due to the formation of insoluble zinc complexes inside the synaptosomes. The results are discussed in terms of the modulation of diverse neurochemical processes by zinc.  相似文献   

19.
Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.  相似文献   

20.
A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号