scholarly journals Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts.

1986 ◽  
Vol 87 (1) ◽  
pp. 47-72 ◽  
Author(s):  
B G Kennedy ◽  
G Lunn ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Conformational Change ◽  
Red Blood Cell ◽  
Forward Direction ◽  
Nucleotide Composition ◽  
Reaction Cycle ◽  
High Affinity ◽  
High Affinity Site ◽  
Na Efflux ◽  
External K

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.

scholarly journals Comparison of the side-dependent effects of Na and K on orthophosphate-, UTP-, and ATP-promoted ouabain binding to reconstituted human red blood cell ghosts.

1976 ◽  
Vol 67 (5) ◽  
pp. 527-545 ◽  
Author(s):  
H H Bodemann ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Adenosine Triphosphate ◽  
Mechanism Of Action ◽  
Red Blood Cell ◽  
Ouabain Binding ◽  
Uridine Triphosphate ◽  
Binding Rate ◽  
External K

This paper is concerned with analyzing the sidedness of action of various determinants which alter the rate of ouabain binding to human red blood cell ghosts. Thus, ouabain binding promoted by orthophosphate (Pi) and its inhibition by Na are shown to be due to inside Pi and inside Na. External K inhibits Pi-promoted ouabain binding and Nao acts to decrease the effectiveness of Ko. Similarly, inside uridine triphosphate (UTPi) stimulates the rate of ouabain binding which can be antagonized by either Nai or Ko acting alone. The actions of Nai and Ko are different when ouabain binding is promoted by Pi and UTPi compared to inside adenosine triphosphate (ATPi). With ATPi, the ouabain binding rate is only affected when Nai and Ko are both present. Possible differences in the mechanism of action of K and Na on Pi-and UTP-promoted binding are discussed in the light of their sidedness of action.

scholarly journals Ion–Ion Interactions at the Selectivity Filter

2000 ◽  
Vol 115 (4) ◽  
pp. 509-518 ◽  
Author(s):  
David Immke ◽  
Stephen J. Korn
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Selectivity Filter ◽  
Na Current ◽  
High Affinity ◽  
Ion Interactions ◽  
High Affinity Site ◽  
Maximal Block ◽  
Different Levels ◽  
External K

In the Kv2.1 potassium channel, binding of K+ to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na+ current, K+ interacts with the TEA modulation site at concentrations ≤30 μM. In this paper, we further characterized the TEA modulation site and examined how varying K+ occupancy of the pore influenced the interaction of K+ with this site. In the presence of high internal and external [K+], TEA blocked 100% of current with an IC50 of 1.9 ± 0.2 mM. In the absence of a substitute permeating ion, such as Na+, reducing access of K+ to the pore resulted in a reduction of TEA efficacy, but produced little or no change in TEA potency (under conditions in which maximal block by TEA was just 32%, the IC50 for block was 2.0 ± 0.6 mM). The all-or-none nature of TEA block (channels were either completely sensitive or completely insensitive), indicated that one selectivity filter binding site must be occupied for TEA sensitivity, and that one selectivity filter binding site is not involved in modulating TEA sensitivity. At three different levels of K+ occupancy, achieved by manipulating access of internal K+ to the pore, elevation of external [K+] shifted channels from a TEA-insensitive to -sensitive state with an EC50 of ∼10 mM. Combined with previous results, these data demonstrate that the TEA modulation site has a high affinity for K+ when only one K+ is in the pore and a low affinity for K+ when the pore is already occupied by K+. These results indicate that ion–ion interactions occur at the selectivity filter. These results also suggest that the selectivity filter is the site of at least one low affinity modulatory effect of external K+, and that the selectivity filter K+ binding sites are not functionally interchangeable.

High affinity interactions between red blood cell receptors and synthetic Plasmodium thrombospondin-related apical merozoite protein (PTRAMP) peptides

Biochimie ◽  
2008 ◽  
Vol 90 (5) ◽  
pp. 802-810 ◽  
Author(s):  
Juan Carlos Calderón ◽  
Hernando Curtidor ◽  
Oscar González ◽  
Gladys Cifuentes ◽  
Claudia Reyes ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Cell Receptors ◽  
High Affinity

scholarly journals Interaction of External K, Na, and Cardioactive Steroids with the Na-K Pump of the Human Red Blood Cell

1974 ◽  
Vol 63 (2) ◽  
pp. 123-143 ◽  
Author(s):  
John R. Sachs
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Ouabain Binding ◽  
Long Period ◽  
Steady State Kinetic ◽  
Straight Line ◽  
Cardioactive Steroids ◽  
Simple Kinetic Model ◽  
State Kinetic ◽  
External K

The interaction of extracellular Na (Nao), K (Ko), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Nao, Ko, and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K1/2 of the pump for Ko, but does not change the form of the relation between the reciprocal of the active K influx (iMKP–1) and the reciprocal of [Ko] ([Ko]–1); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between iMKP–1 vs. [Ko]–1; the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Nao. The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Nao is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low Ko than with strophanthidin.

scholarly journals Inhibition of the Na,K pump by vanadate in high-Na solutions. Modification of the reaction mechanism by external Na acting at a high-affinity site.

1987 ◽  
Vol 90 (2) ◽  
pp. 291-320 ◽  
Author(s):  
J R Sachs
Keyword(s):  
Reaction Mechanism ◽  
Protective Effect ◽  
High Affinity ◽  
High Affinity Site ◽  
Vanadate Inhibition ◽  
External K

We have examined vanadate inhibition of the Na,K pump in the presence of external Na (Nao). Nao protects against inhibition of the Na,K pump by vanadate, but not against inhibition by phosphate or arsenate. Protection by Nao is reversed by external K (Ko). Although the site at which Na exerts its protective effect has properties similar to the two transport sites for K at the outside of the pump, it is not one of the transport sites. The data can be qualitatively accounted for if it is postulated that there is a protective site, separate from the transport sites, at which Nao and Ko compete. When the site is empty or bound to K, vanadate combines with high affinity with pumps that have two K ions bound to the transport sites, but not with pumps that have Na bound to the protective site, even if K is bound to the transport sites. The protective site has a high affinity for both Na and K; the apparent K 1/2 for external Na is less than 2 mM, which is similar to that of a previously described site at which Nao inhibits a number of the partial reactions of the pump. Nao protects against vanadate inhibition of the K-K exchange in the absence of cell Na, and against vanadate inhibition of p-nitrophenylphosphatase activity of the pump in the absence of ATP. The protective site is a manifestation of an E2 conformation of the pump. The protective effect of Nao is not changed by altering the intracellular Mg2+ concentration.

scholarly journals A Ca2+-stimulated adenosine triphosphatase in Golgi-enriched membranes of lactating murine mammary tissue

1984 ◽  
Vol 224 (1) ◽  
pp. 39-45 ◽  
Author(s):  
C D Watters
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Mammary Tissue ◽  
Marker Enzyme ◽  
High Affinity ◽  
Cell Plasma ◽  
Lactating Mammary Gland

A membrane fraction isolated from lactating murine mammary tissue and enriched for the Golgi membrane marker enzyme galactosyltransferase exhibited Ca2+-stimulated ATPase activity (Ca-ATPase) in 20 microM-free Mg2+ and 10 microM-MgATP, with an apparent Km for Ca2+ of 0.8 microM. Exogenous calmodulin did not enhance Ca2+ stimulation, nor could Ca-ATPase activities be detected in millimolar total Mg2+ and ATP. When assayed with micromolar Mg2+ and MgATP the Ca-ATPases of skeletal-muscle sarcoplasmic reticulum and of calmodulin-enriched red blood cell plasma membranes were half-maximally activated by 0.1 microM- and 0.6 microM-Ca2+ respectively. All three Ca-ATPases were inhibited by similar micromolar concentrations of trifluoperazine, but the Golgi activity was unaffected by quercetin in concentrations which completely inhibited both the sarcoplasmic-reticulum and red-blood-cell enzymes. The results are consistent with the hypothesis that the high-affinity Ca-ATPase is responsible for the ATP-dependent Ca2+ transport exhibited by Golgi-enriched vesicles derived from lactating mammary gland [Neville, Selker, Semple & Watters (1981) J. Membr. Biol. 61, 97-105; West (1981) Biochim. Biophys. Acta 673, 374-386].

scholarly journals ADP+orthophosphate (P(i)) stimulates an Na/K pump-mediated coefflux of P(i) and Na in human red blood cell ghosts.

1994 ◽  
Vol 104 (1) ◽  
pp. 33-55 ◽  
Author(s):  
R Marín ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Red Blood Cells ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Dependent Manner ◽  
Red Cell ◽  
Human Red Blood Cells ◽  
Mode Of Operation ◽  
Na Efflux

The Na/K pump in human red blood cells that normally exchanges 3 Nai for 2 Ko is known to continue to transport Na in a ouabain-sensitive and ATP-dependent manner when the medium is made free of both Nao and Ko. Although this Na efflux is called "uncoupled" because of removal of ions to exchange with, the efflux has been shown to be comprised of a coefflux with cellular anions. The work described in this paper presents a new mode of operation of uncoupled Na efflux. This new mode not only depends upon the combined presence of ADP and intracellular orthophosphate (P(i))i but the Na efflux that is stimulated to occur is coeffluxed with (P(i))i. These studies were carried out with DIDS-treated resealed red cell ghosts, suspended in buffered (NMG)2SO4, that were made to contain, in addition to other constituents, varying concentrations of ADP and P(i) together with Na2 SO4, MgSO4 and hexokinase. While neither ADP nor P(i) was effective alone, ouabain-sensitive uncoupled Na efflux, (measured with 22Na) could be activated by [ADP+P(i)] where the K0.5 for ADP in the presence of 10 mmol (P(i))i/liter ghosts was 100-200 mumol/liter ghosts and the K0.5 for (P(i))i, in the presence of 500 mumol ADP/liter ghosts was 3-4 mmol/liter ghosts. [ADP+P(i)] activation of this Na efflux could be inhibited by as little as 2 mumol ATP/liter ghosts but the inhibition could be relieved by the addition of 50 mM glucose, given entrapped hexokinase. While ouabain-sensitive Na efflux was found to be coeffluxed with P(i) (measured with entrapped [32P]H3PO4), this was not so for SO4 (measured with 35SO4). The stoichiometry of Na to P(i) efflux was found to be approximately 2 to 1. Na efflux as well as (P(i))i efflux were both inhibited by 10 mM Nao (K0.5 approximately equal to 4 mM). But, whereas 20 mM Ko (K0.5 approximately equal to 6 mM) inhibited the efflux of (P(i))i, as would be expected from previous work, Na efflux was actually increased. When Ko influx was measured in this situation there was a 1 for 1 exchange of Nai for Ko, that is, of course, downhill with respect to the gradient of each ion. Surprisingly AsO4 was unable to replace P(i) for activation of Na efflux but Na efflux could be inhibited by vanadate and oligomycin. In terms of mechanism, it is likely that ADP acts to promote the formation of the phosphoenzyme (EP) by (P(i))i that would otherwise be inhibited by Nai.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interaction between phloretin and the red blood cell membrane.

1976 ◽  
Vol 67 (4) ◽  
pp. 381-397 ◽  
Author(s):  
M L Jennings ◽  
A K Solomon
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Binding Sites ◽  
Time Course ◽  
Transport Processes ◽  
Red Cells ◽  
Affinity Binding ◽  
Red Cell ◽  
High Affinity

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.

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