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1.
The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using 86Rb as a K analog, we have measured the rate constant of 86Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes. 相似文献
2.
《生物化学与生物物理学报:生物膜》1986,856(2):408-411
The effects of variations of the NAD+/NADH quotient on the uptake of 86Rb by human red cells loaded by non-disruptive means with the chelator Benz2 and different amounts of 45Ca has been examined. The NAD+/NADH quotient was modified by the addition of pyruvate and/or lactate or xylitol. It was found that the uptake of 86Rb at a given intracellular Ca2+ concentration was faster in the reduced state (lactate or xylitol added). Metabolic changes were associated with variations of the redox state. However, glycolitic intermediates did not significantly modify the apparent affinity for Ca2+ of the Ca2+-dependent K+ channel in one-step inside-out vesicles prepared from the erythrocyte membrane. Taken together, these results suggest that modifications of the cytoplasmic redox potential could modulate the sensitivity to Ca2+ of the Ca2+-dependent K+ channel in the human red cells under physiological conditions. This conclusion is consistent with previous findings in inside-out vesicles of human erythrocytes using artificial electron donors. 相似文献
3.
Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes 总被引:2,自引:0,他引:2
Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD. 相似文献
4.
RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes. 总被引:4,自引:0,他引:4
R Sharma S S Singhal J Cheng Y Yang A Sharma P Zimniak S Awasthi Y C Awasthi 《Archives of biochemistry and biophysics》2001,391(2):171-179
We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes. 相似文献
5.
S. N. Orlov E. J. Cragoe Jr. O. Hänninen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(2):135-140
β-Adrenergic- and volume-dependent regulation of 22Na influx and 86Rb influx and efflux in erythrocytes of brown trout (Salmo trutta m. lacustris) were studied. Norepinephrine (10-6 mol·1-1) increased the rate of 22Na influx 10-to 20-fold via the activation of a Na/H exchanger (ethyl isopropyl amiloride inhibited component of 22Na influx). Unlike carp erythrocytes the activity of the Na, K-pump (ouabain-inhibited 86Rb influx) was only slightly (25–35%) increased by norepinephrine. The norepinephrine-induced increment of Na, K-pump activity
was completely abolished by ethyl isopropyl amiloride thus indicating that this effect was mediated by Na/H exchanger-induced
increase of intracellular Na+ concentration. Cell shrinkage in hyperosmotic media resulted in a several-fold activation of the Na/H exchanger. Cell swelling
in hypotonic media increased both the rate of K, Cl-cotransport [((dihydroindenyl)oxy)alcanaic acidsensitive components of
86Rb influxe and efflux] and passive permeability (leakage) of erythrocyte membranes for Na+ and K+. No volume-dependent regulation of Na, K, 2Cl-cotransport (bumetanide-sensitive components of 86Rb fluxes) was found. It may be concluded that the regulation of monovalent cation transport in erythrocytes of fast-moving
(carnivorous) brown trout differs essentially from that in slowly moving (herbivorous) carp. 相似文献
6.
《生物化学与生物物理学报:生物膜》1986,860(1):25-34
Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca2+-induced loss of KCl from human red cells. However, the Ki values for this effect did not bear good correlation with the Ki values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KCl fluxes were also affected. Added calmodulin did not have any effect on Ca2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported Ki for calmodulin-dependent systems. These results suggest that Ca2+-dependent K+ channels of red cells are not regulated by calmodulin. 相似文献
7.
Wesołowska O Mosiadz D Motohashi N Kawase M Michalak K 《Biochimica et biophysica acta》2005,1720(1-2):52-58
The expression of multidrug resistance-associated protein (MRP1) results in ATP-dependent reduction of drugs' concentration in cancer cells, i.e., multidrug resistance (MDR). Since the majority of projects are concentrated on the search of the new MDR modulators, there are very few reports on drug-induced stimulation of MDR transporters activity. In the present work, by means of functional fluorescence assay we have shown that MRP1-mediated efflux of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by phenothiazine maleates that have been already identified as P-glycoprotein inhibitors. Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2',7'-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes has been also demonstrated. Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. To our best knowledge, this report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. We conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site. 相似文献
8.
Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of 32P-spectrin with inside-out vesicles with a Ki of 10?7M, and causes rapid dissociation of 32P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte. 相似文献
9.
Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes 总被引:1,自引:0,他引:1
E F LaBelle S V Singh S K Srivastava Y C Awasthi 《Biochemical and biophysical research communications》1986,139(2):538-544
The effect of oxidized glutathione (GSSG) on the ATP-dependent transport of S-dinitrophenyl glutathione (Dnp-SG) by inside-out vesicles prepared from human erythrocytes and by intact erythrocytes has been studied. It is demonstrated that the transport of Dnp-SG is not inhibited by GSSG in either intact erythrocytes or in inside-out vesicles. These results suggest that Dnp-SG and GSSG are transported out of human erythrocytes by separate systems. 相似文献
10.
Małgorzata Bobrowska-Hägerstrand Anna Wróbel Błazej Rychlik Ida Öhman 《Molecular membrane biology》2013,30(5-6):485-495
The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants Km and Vmax of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry. 相似文献
11.
12.
Summary The plasma membrane (Mg2+)-dependent adenosine triphosphatase ((Mg2+)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg2+)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H+ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca2+)-dependent adenosine triphosphatase ((Ca2+)-ATPase) activity. Proton efflux associated with (Ca2+)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca2+)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem.
257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg2+)-ATPase to pump28Mg2+,35SO
4
2–
and86Rb+ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system. 相似文献
13.
Rashid H Ali MK Tayyab S 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2000,127(3):345-350
Interaction of bilirubin with different types of erythrocyte membrane vesicles such as unsealed, heterogeneous, sealed and inside-out membrane vesicles prepared from human and goat erythrocytes was studied. Out of various types of membrane vesicles, in both species, unsealed membrane vesicles bound quantitatively higher amounts of bilirubin followed by heterogeneous and sealed membrane vesicles whereas inside-out membrane vesicles bound the lowest amount of bilirubin. These differences in the amount of bound bilirubin to different membrane vesicles were correlated well with the percentage accessibility of sialic acid to neuraminidase in these membranes suggesting that bilirubin bound preferentially to the outer layer of erythrocyte membranes than the inner layer. Further, membrane vesicles prepared from human erythrocytes bound higher amounts of bilirubin than those prepared from goat erythrocytes. This can be ascribed to different phospholipid composition of these membranes. 相似文献
14.
In this work we show the existence of cyclic AMP phosphodiesterase (EC 3.1.4.17) in human erythrocyte membranes and have clarified some properties of the enzyme. In human erythrocytes, about 23% of the total cyclic AMP phosphodiesterase activity is in a membrane-bound form. Although it could be solubilized with Triton X-100 in 5 mM Tris-HCl buffer (pH 8.0), it was not solubilized by a low or high concentration of salt. The enzyme seems to be localized in the cytoplasmic surface, since it is detected in sealed inside-out vesicles of human erythrocyte membranes, but not in intact human erythrocytes. The optimum pH was found to lie between 7.4 and 8.0, and Mg2+ was found to be necessary for its activity. Ca2+ and calmodulin could not stimulate the activity of this enzyme. Theophylline was a strong inhibitor, but cyclic GMP could not inhibit the enzymic hydrolysis of cyclic [32P]AMP and this membrane-bound enzyme therefore seems to be specific to cyclic AMP. 相似文献
15.
Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes 总被引:8,自引:3,他引:5
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The relationship between active extrusion of Ca++ from red cell ghosts and active uptake of Ca++ by isolated red cell membrane fragments was investigated by studying the Ca++ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca++ adenosine triphosphatase (ATPase) and ATP-dependent Ca++ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more 45Ca in the presence of ATP compared to preparation B. Separation of the 45Ca-labeled membrane vesicles by density gradient centrifugation showed that the 45Ca label was localized to the inside-out vesicle fraction. In addition, the 45Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of 45Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La+8 (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca++ by red cell membrane fragments is due to the active transport of Ca++ into inside-out vesicles. 相似文献
16.
Proton-activated rubidium transport catalyzed by the sodium pump 总被引:1,自引:0,他引:1
R Blostein 《The Journal of biological chemistry》1985,260(2):829-833
Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions. 相似文献
17.
1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. Incubation of vesicles with [gamma-32P]ATP at 37 degrees C for periods of up to 1 hr did not change the topography or the permeability of the vesicles. 3. Vesicles incorporated labeled phosphate from [gamma-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side out vesicles were not as active. 5. Impermeable right-side-out vesicles, treated with 0.01 percent saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane. 相似文献
18.
Erythrocyte and HeLa cell plasma membranes were isolated on polylysinecoated polyacrylamide beads and the transbilayer disposition of their proteins was investigated.When membranes of intact erythrocytes were isolated on beads and then labelled by lactoperoxidase-catalysed iodination, their labelling pattern was similar to that of inside-out vesicles in solution.When the membranes of intact HeLa cells were isolated on beads and then labelled by galactose oxidase-[3H]borohydride treatment, no glycoprotein or glycolipid sugars were accessible. On the other hand, when the HeLa cell membranes were isolated on beads and then labelled by the lactoperoxidase-catalysed iodination, all of the major membrane proteins were iodinated. These experiments confirmed for HeLa cell membranes what had previously been shown for erythrocyte membranes: when the membranes of intact cells are isolated on beads, the accessibility of their surfaces to enzymatic probes is the same as would be expected of inside-out vesicles in suspension. Double-label experiments, in which the HeLa cell membranes were labelled first on the intact HeLa cells and again after isolation on beads, identified several 相似文献
19.
Summary Preparations of avian erythrocyte plasma membranes have been made which are in the form of sealed vesicles. Using these preparations the permeability of the membranes to Na+ K+, Mg+ and Ca+ was measured. Monobutyryl cyclic AMP and cyclic AMP increased the permeability to Na+ and Ca+ under conditions where no protein phosphorylation could occur. The only effect of phosphorylation of membrane proteins was to reduce Ca+ permeability. It is thus concluded that cyclic AMP increases Na+ permeability in the avian erythrocyte by a direct effect which does not involve protein phosphorylation. 相似文献
20.
The following evidence supports the concept that cholesterol in membranes is the receptor and target site for the cytolytic action of cereolysin. (i) Of the various phospholipids, gangliosides, and steroids tested, only cholesterol and closely related sterols (sitosterol and dihydrocholesterol) significantly inhibited the hemolytic activity of cereolysin. (ii) Acholeplasma laidlawii cells grown in the presence of cholesterol inhibited the hemolytic activity of cereolysin, but A. laidlawii grown in the absence of cholesterol did not. (iii) Incubation of A. laidlawii cells, grown in the absence of cholesterol, with a cholesterol-Tween 80 mixture reestablished the ability of the cells to bind cereolysin. (iv) Treatment of erythrocyte membranes and A. laidlawii cells containing cholesterol with cholesterol oxidase (EC 1.1.3.6, Brevibacterium species) abolished the ability of these membranes to bind cereolysin and inhibit the hemolytic activity of the toxin, (v) Cereolysin could bind to and alter the permeability of both right-side-out ghosts and inside-out vesicles prepared from human erythrocytes, in agreement with other data that cholesterol is present on both sides of the erythrocyte membrane, (vi) Cereolysin caused the release of [14C]glucose from liposomes containing cholesterol, and this release was dependent on the amount of cholesterol in the liposomes. 相似文献