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1.
Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells 总被引:6,自引:3,他引:3
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Philip A. Knauf Fulgencio Proverbio Joseph F. Hoffman 《The Journal of general physiology》1974,63(3):305-323
The phosphoproteins formed by incubation of red cell ghosts with [γ-32P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The 32P-labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of 32P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the 32P-phosphoprotein present in the gels. The molecular weight of the 32P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na:K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts. 相似文献
2.
Helmut Weicker Wilfried Bieger Helga Huster 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,583(2):189-195
The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K+-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na+ + K+)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K+-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl3 at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells. 相似文献
3.
Sodium movements in the human red blood cell 总被引:19,自引:9,他引:10
J R Sachs 《The Journal of general physiology》1970,56(3):322-341
Measurements were made of the sodium outflux rate constant, o
k
Na, and sodium influx rate constant, i
k
Na, at varying concentrations of extracellular (Nao) and intracellular (Nac) sodium. o
k
Na increases with increasing [Nao] in the presence of extracellular potassium (Ko) and in solutions containing ouabain. In K-free solutions which do not contain ouabain, o
k
Na falls as [Nao] rises from 0 to 6 mM; above 6 mM, o
k
Na increases with increasing [Nao]. Part of the Na outflux which occurs in solutions free of Na and K disappears when the cells are starved or when the measurements are made in solutions containing ouabain. As [Nao] increases from 0 to 6 mM, i
k
Na decreases, suggesting that sites involved in the sodium influx are becoming saturated. As [Nac] increases, o
k
Na at first increases and then decreases; this relation between o
k
Na and [Nac] is found when the measurements are made in high Na, high K solutions; high Na, K-free solutions; and in (Na + K)-free solutions. The relation may be the consequence of the requirement that more than one Na ion must react with the transport mechanism at the inner surface of the membrane before transport occurs. Further evidence has been obtained that the ouabain-inhibited Na outflux and Na influx in K-free solutions represent an exchange of Nac for Nao via the Na-K pump mechanism. 相似文献
4.
The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K+-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na+ + K+)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K+-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl3 at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells. 相似文献
5.
Summary
3H-labelled phloretin was shown to be bound reversibly by human erythrocyte and ghost membranes but not to penetrate across them in either direction. Kinetic parameters ofd-xylose andd-galactose transport in intact cells and in ghosts, as well as the inhibition by phloretin of these transports were found to be in fair agreement. By enclosing phloretin in ghosts, its inhibition of monosaccharide transport was found to be symmetrical and thus an equivalence of the outer and the inner membrane sides of the human erythrocyte was demonstrated. 相似文献
6.
Robert Nielsen 《The Journal of membrane biology》1979,51(2):161-184
Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K+ because filipinper se increases the nonspecific Na+ and K+ permeability of the outward facing membrane. The K+ transport was calculated from the chemically determined changes in K+ concentrations in the solution bathing the two sides of the skin. The active transepithelial K+ transport required the presence of Na+ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na+, K+-ATPase inhibitor ouabain. The addition of Ba++ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K+ transport and in an inhibition of the active Na+ transport. This is in agreement with the notion that Ba++ decreases the passive K+ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba++ there was a good correlation between the active Na+ and K+ transport. It is concluded that the active transepithelial K+ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5. 相似文献
7.
K P Wheeler 《The Biochemical journal》1982,202(3):613-621
Resealed ghosts from pigeon erythrocytes were shown to haemolyse during incubation in isotonic media with pH values greater than about 7 and high concentrations of Na+ inside the ghosts seemed to enhance this effect. At lower pH values the ghosts were stable but still highly permeable to Na+ and K+, and moderately permeable to sucrose. Under the latter conditions the ghosts transported amino acids in a way qualitatively but not quantitatively similar to intact erythrocytes. The Na+-dependent transport of serine and alanine by the ghosts consisted essentially of an exchange of extracellular for intracellular amino acids, with no significant net flux. In contrast, net fluxes of glycine in the direction of the Na+-concentration gradient across the ghost membrane were demonstrated. However, under one condition a small net influx of glycine occurred against the prevailing Na+-concentration gradient. Unlike Na+-dependent glycine uptake, the uptake of six other amino acids by intact pigeon erythrocytes was not influenced by the nature of the anion present. The significance of these findings in relation to previous work on the Na+-gradient hypothesis of membrane transport is discussed. 相似文献
8.
TEORELL T 《The Journal of general physiology》1952,35(5):669-701
1. Erythrocyte ghosts from human blood were produced by gentle water hemolysis. The ghost-containing hemolysate (about 20 mN) was added to media of different composition (KCl, NaCl, glucose, sucrose, etc.) and varying concentration ranging from 8 to 840 mN. The volume changes of the ghost cells were followed by a light absorption method. The potassium and sodium concentrations were also analyzed in some representative cases. 2. The ghosts shrank, or swelled, in two stages. An initial phase with a momentary expulsion, or uptake, of water leading to an osmotic equilibrium, was followed by a second phase in which a slow swelling or shrinking proceeded toward a final constant volume. 3. The ghosts were semipermeable in the sense that water always passed rapidly in either direction so as to maintain isotonicity with the external medium. The relation between ghost cell volumes (V) and the total concentration (C(e)) of the suspension medium can be expressed by a modified van't Hoff-Mariotte law: (C(e) + a)(V - b) = constant. Here a is a term correcting for an internal pressure and b is the non-solvent volume of the ghost cells. This means that the ghosts behave as perfect osmometers. 4. On the other hand appreciable concentration differences of the K and Na ions could be maintained across the intact ghost cell membranes for long periods. Whether this phenomenon is due simply to very low cation permeability or to active transport processes cannot be decided, although the first assumption appears more probable. 5. When the ghosts were treated with small concentrations of a lytic substance like Na oleate, the alkali ion transfer was greatly increased. This seems to be a simple exchange diffusion process with simultaneous, continued maintenance of osmotic equilibrium (= the second phase). A simplified theory is also given for the kinetics of the volume variations and ion exchange during the second phase (cf. the Appendix). 6. Miscellaneous observations on the effects of pH, and of some other substances are discussed. Some shape transformations of the ghost cells are also described. 相似文献
9.
Hyun Dju Kim Martha Baird John Sallis Stewart Nicol Russell E. Isaacks 《Biochemical and biophysical research communications》1984,119(3):1161-1167
The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb86 influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb86 influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes. 相似文献
10.
Ouabain-Insensitive Sodium Movements in the Human Red Blood Cell 总被引:15,自引:6,他引:9
John R. Sachs 《The Journal of general physiology》1971,57(3):259-282
Red blood cells exposed to ouabain are capable of net Na outflux against an electrochemical gradient; the net outflux is inhibited by the diuretic, furosemide. In ouabain-treated cells, both the unidirectional Na outflux and the unidirectional Na influx are inhibited by furosemide. Furosemide also inhibits the ouabain-sensitive Na-Na exchange accomplished by the Na-K pump in K-free solutions. From the interaction of extracellular K, furosemide, and ouabain with the transport system, it seems possible that the ouabain-insensitive Na outflux is accomplished by the same mechanism that is responsible for the ouabain-sensitive Na-K exchange. The ouabain-insensitive Na outflux is increased by extracellular Na, and the influx increases as the intracellular Na increases. In fresh cells, high extracellular K concentrations decrease the ouabain-insensitive Na outflux and increase the ouabain-insensitive Na influx. When the rate constant for sodium outflux and the rate constant for sodium influx in ouabain-treated cells are plotted against the extracellular K concentration, the curves obtained are mirror images of each other. In starved cells, extracellular K increases the ouabain-insensitive Na outflux as does extracellular Na, and it has little effect on the Na influx. 相似文献
11.
Sodium Transport in Capillaries Isolated from Rat Brain 总被引:20,自引:12,他引:8
A. Lorris Betz 《Journal of neurochemistry》1983,41(4):1150-1157
Abstract: Brain capillary endothelial cells form a bloodbrain barrier (BBB) that appears to play a role in fluid and ion homeostasis in brain. One important transport system that may be involved in this regulatory function is the Na+,K+-ATPase that was previously demonstrated to be present in isolated brain capillaries. The goal of the present study was to identify additional Na+ transport systems in brain capillaries that might contribute to BBB function. Microvessels were isolated from rat brains and 22Na + uptake by and efflux from the cells were studied. Total 22Na + uptake was increased and the rate of 22Na + efflux was decreased by ouabain, confirming the presence of Na+,K+-ATPase in capillary cells. After inhibition of Na+,K+-ATPase activity, another saturable Na + transport mechanism became apparent. Capillary uptake of 22Na + was stimulated by an elevated concentration of Na +or H+ inside the cells and inhibited by extracellular Na+, H+, Li+, and NH4+. Amiloride inhibited 22Na + uptake with a Ki between 10?5 and 10?6M but there was no effect of 1 mM furosemide on 22Na+ uptake by the isolated microvessels. These results indicate the presence in brain capillaries of a transport system capable of mediating Na +/ Na + and Na +/H + exchange. As a similar transport system does not appear to be present on the luminal membrane of the brain capillary endothelial cell, it is proposed that Na +/H + exchange occurs primarily across the antiluminal membrane. 相似文献
12.
Oscar A. Candia 《Biophysical journal》1970,10(4):323-344
Excitability (action potential and refractory period) has been described by A. Finkelstein in the depolarizing region of the current-voltage (I-V) curve of the isolated frog skin. Recently Fishman and Macey interpreted this phenomenon as a consequence of a region with negative resistance that confers to the I-V curve an N shape. We have studied the I-V relation of the isolated frog skin in the hyperpolarizing region with a current-ramp system. It was found that in Na2SO4 Ringer's, the resistance continuously increases in the hyperpolarizing direction. When hyperpolarization reaches 300 mv an electrical breakdown occurs, occasionally followed by a region of negative resistance. In NaCl Ringer's the breakdown was also found although the I-V relation was reasonably linear. Unidirectional Na+ outflux was measured at different levels of voltage clamping across the skin and with different Na+ concentrations in the solutions. The Na+ outflux was found to be relatively independent of these parameters. Based on these results a Na+ rectifying structure is postulated. An electrical model for active Na+ transport including a diode and an oscillator is proposed. The effects of CO2, nitrogen, amiloride, and ouabain on the I-V relation are described. 相似文献
13.
Guillermo Whittembury 《The Journal of general physiology》1965,48(4):699-717
Slices from the cortex corticis of the guinea pig kidney were immersed in a chilled solution without K and then reimmersed in warmer solutions. The Na and K concentrations and the membrane potential Vm were then studied as a function of the Na and K concentrations of the reimmersion fluid. It was found that Na is extruded from the cells against a large electrochemical potential gradient. Q10 for net Na outflux was ∼2.5. At bath K concentrations larger than 8 mM the behavior of K was largely passive. At the outset of reimmersion (Vm > EK) K influx seemed secondary to Na extrusion. Na extrusion would promote K entrance, being limited and requiring the presence of K in the bathing fluid. At bath K concentrations below 8 mM, K influx was up an electrochemical potential gradient. Thus a parallel active K uptake is apparent. Q10 for net K influx was ∼2.0. Dinitrophenol inhibited net Na outflux and net K influx, Q10 became <1.1 for both fluxes. The ratio between these fluxes varied. Thus at the outset of reimmersion the net Na outflux to net K influx ratio was >1. After 8 minutes it was <1. 相似文献
14.
The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant
(R) rats. Previous reports have suggested that there is a difference in primary sequence in the α1 subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion
in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps
isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the
Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers
cultured from S rats transport about twice as much Na+ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of
the affinities for ouabain, Na+, and K+, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the
number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na+ transport rates. The analysis of the steady-state Na+ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity
than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of
Na+ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na+. They suggest that the epithelial Na+ channel is intrinsically different or differently regulated in S and R rats.
Received: 6 May 1996/Revised: 16 October 1996 相似文献
15.
Julia E. Lever 《Journal of cellular biochemistry》1977,6(1):103-124
Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na+ gradient, external Na+ > internal Na+, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na+-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na+-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent Km values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na+ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na+ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na+, K+-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na+. The apparent Km for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na+ concentration. Similarly, in the presence of a standard initial Na+ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K+ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na+ and was blocked by the further addition of either nigericin or external K+. As a corollary, Na+-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na+-coupled active transport of these amino acids but did not affect Na+-independent transport. Translocation of K+, H+, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na+ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na+, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na+-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na+ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed. 相似文献
16.
Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na+ were found to take up Ca2+ when incubated in K+ media or in sucrose media containing micromolar concentrations of free Ca2+. Li+- or choline+loaded ghosts did not take up Ca2+. The Ca2+ accumulated by Na+-loaded ghosts could be released by the Ca2+ ionophore A23187, but not by EGTA. Ca2+ uptake was inhibited by external Sr2+, Na +, Li +, or choline +. All the 45Ca2+ accumulated by Na+-dependent Ca2+ uptake could be released by external Na +, indicating that both Ca2+ influx and efflux occur in a Na+-dependent manner. Na + -dependent Ca2+ uptake and release were only slightly inhibited by Mg2+. In the presence of the Na+ ionophore Monensin the Ca2+ uptake by Na +-loaded ghosts was reduced. Ca2+ sequestered by the Na+-dependent mechanism could also be released by external Ca2+ or Sr2+ but not by Mg2+, indicating the presence of a Ca2+/Ca2+ exchange activity in secretory membrane vesicles. This Ca2+/Ca2+ exchange system is inhibited by Mg2+, but not by Sr2+. The Na + -dependent Ca2+ uptake system in the presence of Mg2+ is a saturable process with an apparent Km of 0.28 μM and a Vmax= 14.5 nmol min?1 mg protein?1. Ruthenium red inhibited neither the Na+/Ca2+ nor the Ca2+/Ca2+ exchange, even at high concentrations. 相似文献
17.
Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts 总被引:1,自引:0,他引:1
In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells. 相似文献
18.
Our preliminary studies have shown that the Na,K-pump in frog erythrocytes is activated by isoproterenol (ISP), phosphodiesterase
blocker (3-isobutyl-methylxantine, IBMX), and by iodoacetate (MIA). The aim of the present study was to determine a mechanism
responsible for the effect of MIA on the Na,K-pump activity in frog red blood cells as well as the role of G proteins and
intracellular messengers in modulation of active K+ transport induced by ISP. An additive stimulation of active K+ (86Rb) transport in frog erythrocytes was found after exposure of the cells to MIA in a combination with ISP or IBMX. The treatment
of the red blood cells with 1 mM MIA for 1 or 2 h was associated with a significant decrease in intracellular Na+ concentration, on average, by 13 and 20%, respectively, suggesting a direct action of MIA on the Na,K-pump. Incubation of
cells in the presence of dibutyryl-cAMP (1 mM) or adenylate cyclase activator forskolin (0.1 mM) caused stimulation of the
active K+ influx by 21.8 and 27.9%, respectively. AlF
4
-
and cholera toxin able to increase cell cAMP levels via G protein interactions had no effect on the total and IPS-induced
K+ influx in frog erythrocytes. The treatment of the red blood cells with sodium nitroprusside that increases cGMP concentration
in cells also had no effect on the K+ influx. The stimulatory influence of ISP on the Na,K-pump was reduced with increase of the intracellular Na+ concentration. ISP increased affinity of the Na,K-pump to Na+ (the Mihaelis constant KM = 34.4 ± 5.1 in control and 25.3 ± 2.8 mM in the presence of ISP,p < 0.01), but did not change maximal velocity (8.1 ± 0.6 and 7.7 ± 0.3 mmol/1/h in the control and ISP-treated cells, respectively).
The results obtained indicate the presence of several different signal pathways involved in regulation of the Na,K-pump activity
in frog erythrocytes. 相似文献
19.
The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins 总被引:2,自引:0,他引:2
Summary The hemolytic activity of the terminal complement proteins (C5b-9) towards erythrocytes containing high potassium concentration has been reported to be dramatically increased when extracellular Na+ is substituted isotonically by K+ (Dalmasso, A.P., et al., 1975,J. Immunol.
115:63–68). This phenomenon was now further investigated using resealed human erythrocyte ghosts (ghosts), which can be maintained at a nonlytic osmotic steady state subsequent to C5b-9 binding: (1) The functional state of C5b-9-treated ghosts was studied from their ability to retain trapped [14C]-sucrose or [3H]-inulin when suspended either in the presence of Na+ or K+. A dramatic increase in the permeability of the ghost membrane to both nonelectrolytes-in the absence of significant hemoglobin release-was observed for C5b-9 assembly in the presence of external K+. (2) The physical binding of the individual125I-labeled terminal complement proteins to ghost membranes was directly measured as a function of intra- and extracellular K+ and Na+. The uptake of125I-C7,125I-C8, and125I-C9 into membrane C5b-9 was unaltered by substitution of Na+ by K+. (3) The binding of the terminal complement proteins to ghosts subjected to a transient membrane potential generated by the K+-ionophore valinomycin (in the presence of K+ concentration gradients) was measured. No significant change in membrane binding of any of the C5b-9 proteins was detected under the influence of both depolarizing and hyperpolarizing membrane potentials. It can be concluded that the differential effect of Na+ versus K+ upon the erythrocyte membrane isnot due to an effect upon the binding of the complement proteins to the membraneper se, but upon the functional properties of the assembled C5b-9 pore site. 相似文献
20.
Shi Liu Ron Jacob David Piwnica-Worms Melvyn Lieberman 《Molecular and cellular biochemistry》1989,89(2):147-150
We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cli above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Nai homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Clo to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a
infNa
supi
to its control level and the accompanying hyperpolarization could be blocked by 10–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring Ko (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of aNa was attenuated by 10–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Nai. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells. 相似文献