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J R Sachs

Publications and source records attributed to J R Sachs.

At least 37 records · Page 2Linked to original sources
PubMed1988

Interaction of magnesium with the sodium pump of the human red cell.

1. In broken red cell membranes, Mg2+ inhibition of Na+,K+-adenosine-5'-triphosphatase (Na+,K+-ATPase) activity was partially competitive with MgATP. Mg2+ inhibition of Na+,K+-ATPase activity was uncompetitive with K+ in broken red cell membranes, and Mg2+ inhibition of ouabain-sensitive K+ influx in K+-free resealed ghosts was uncompetitive with external K+. 2. When Na+,K+-ATPase activity was measured at relatively high K+ concentration, Mg2+ inhibition was partially competitive with Na+. Mg2+ inhibition of ouabain-sensitive K+ influx in K+-free resealed ghosts was competitive with cell Na+. Magnesium was a more effective inhibitor of the uncoupled Na+ efflux in low-Na+ ghosts than in high-Na+ ghosts. 3. These findings indicate that Mg2+ inhibition results from combination of the ion with the enzyme form E2K at high intracellular Na+ and K+, and from combination with the form E1 at low intracellular Na+ and K+. 4. In ghosts containing high concentrations of MgPO4, inhibition of the K+-K+ exchange by Mg2+ was more effective at high than at low nucleotide concentrations. At high MgPO4 and low Mg2+ concentration the activity of the exchange increased monotonically with nucleotide concentration, but at a higher Mg2+ concentration, nucleotide activation of the exchange was biphasic: the K+-K+ exchange rate increased, reached a maximum, and then decreased with increasing nucleotide concentration.

Adenosine Triphosphate↗
PubMed1987

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.

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.

Biological Transport↗
PubMed1986

Potassium-potassium exchange as part of the over-all reaction mechanism of the sodium pump of the human red blood cell.

When the efflux components of the Na-K exchange and K-K exchange are measured under identical conditions, the apparent K 1/2 (the concentration of K at which the velocity is half-maximal) for external K of the two processes differ. The discrepancy diminishes when the measurements are made in solutions containing low concentrations of Na, and in these solutions the uncoupled Na efflux is also partially suppressed. It is possible to demonstrate an uncoupled K efflux into solutions free of Na and K. This uncoupled efflux is also partially inhibited by low concentrations of external Na. At high enough concentrations, intracellular Na completely inhibits the K-K exchange. Inhibition of the K-K exchange by cell Na is competitive with cell K, and inhibition of the Na-K exchange by cell K is competitive with cell Na. In each case the characteristics of the competition suggest that both ions competitively interact with sites on the same enzyme form. The Albers-Post model of the Na-K pump reaction mechanism, modified to account for the uncoupled Na and K efflux, accounts in detail for these observations. If the K-K exchange is part of the over-all Na-K exchange, as indicated by the findings, pump models in which Na must add to the pump at the inside before K is released are excluded.

Biological Transport, Active↗
PubMed1986

The order of addition of sodium and release of potassium at the inside of the sodium pump of the human red cell.

1. Inhibition of Na,K-adenosine 5'-triphosphatase (Na,K-ATPase) activity of human red cell membranes by vanadate rapidly reaches a steady-state level and is rapidly reversible. 2. In K-free cells vanadate inhibits the Na-K exchange at low concentrations, the uncoupled Na efflux at higher concentrations, and has little effect on the Na-Na exchange even at high concentrations. Increasing intracellular K concentration increases the sensitivity of the Na-Na exchange to vanadate. 3. Vanadate inhibition of the Na-K exchange is uncompetitive with extracellular K and inhibition of the K-K exchange is partially uncompetitive with intracellular K. Na-Li exchange is less sensitive to vanadate inhibition than Na-K exchange. The results indicate that vanadate inhibits by combining with an enzyme form which occurs between the addition of K at the outside and its release to the inside. 4. Inhibition by vanadate is non-competitive with Na at low adenosine 5'-triphosphate (ATP) concentrations and high concentrations of K. At high ATP and low K, vanadate inhibition becomes partially uncompetitive with Na. At high or at low ATP and in the absence of cell K, inhibition is strictly uncompetitive with cell Na. 5. These results are consistent with a ping-pong model for the reaction mechanism of the Na pump, but they are not consistent with a sequential mechanism or with a branched-chain mechanism in which Na adds after the release of K in one branch and before in the other.

Adenosine Triphosphate↗
PubMed1981

Mechanistic implications of the potassium-potassium exchange carried out by the sodium-potassium pump.

1. The magnitude of the K-K exchange carried out by the Na pump in human red cells was measured as a function of the external K concentration in cells with high and low intracellular K. The apparent K1/2 for external K and the Vm increased by the same proportion when intracellular K was raised. The result suggests that the K-K exchange is part of a ping-pong reaction mechanism. 2. The velocity of the exchange increases monotonically with intracellular phosphate and ATP concentration; neither substrate inhibits at very high concentration. 3. These results seem to require that the enzyme species which carries out the exchange is both phosphorylated and combined with ATP.

Adenosine Triphosphate↗
PubMed1981

Internal potassium stimulates the sodium-potassium pump by increasing cell ATP concentration.

1. Intracellular K increases the ouabain-sensitive Na-K exchange in human red blood cells. Pump rate increases hyperbolically with internal K with a K12 for K of 2.58 m-mole/l. red blood cells. Li also stimulates the pump rate, but with a much higher K12. The stimulation does not result from a change in the affinity of the pump for its substrates Na, K or Mg or for the product, phosphate. 2. The effect of cell K on the Na-Na exchange is biphasic. At low concentrations it decreases the exchange rate but then the exchange increases linearly with cell k concentration. 3. Stimulation of the pump rate by internal K can be demonstrated in reconstituted ghosts but only if the ratio of the volume of cells to that of solution at the time of haemolysis is high. Stimulation is not observed if the ghosts contain an efficient system for rephosphorylating ADP to ATP, such as creatine phosphate and creatine kinase, or if the measurements are made with iodoacetamide which inhibits rephosphorylation of ADP by inhibiting the enzyme glyceraldehyde-3-phosphate dehydrogenase. 4. Cells with low internal K and Li have low ATP concentrations, and ATP increases hyperbolically with internal K or Li with the same K12 as does the pump rate. In cells depleted of substrate intracellular K does not stimulate the pump rate. 5. The effect of K and Li on the pump rate probably does not result from enhanced activity of any of the enzymes below phosphofructokinase in the glycolytic pathway.

Adenosine Triphosphate↗
PubMed1980

The order of release of sodium and addition of potassium in the sodium-potassium pump reaction mechanism.

1. The characteristics of oligomycin inhibition of the Na--K pump of human red cell membranes was investigated. Oligomycin inhibition of the pump was found to be reversible. 2. Inhibition of Na--K ATPase activity was uncompetitive with respect to ATP in broken membrane preparations. In intact cells inhibition was uncompetitive with respect to both intracellular and extracellular Na. 3. Oligomycin did not significantly inhibit the K--K exchange if the cells were Na-free, but if the cells contained a small amount of Na, oligomycin inhibition of the K--K exchange became significant. Taken together with the findings described above, this indicates that oligomycin combines with E1P-Na conformation of the pump and not with any E2 conformation. 4. When measurements are made in solutions high in Na, oligomycin is a non-competitive inhibitor with respect to external K, but in Na-free solutions, oligomycin inhibition is uncompetitive with respect to external K. 5. These findings indicate that, in the normal operation of the pump, Na is released to the outside before K adds.

Adenosine Triphosphatases↗
PubMed1977

Kinetics of the inhibition of the Na-K pump by external sodium.

1. When the ouabain-sensitive K influx or the ouabain-sensitive Cs influx is measured as a function of the extracellular concentration of K or Cs in Na-free solutions the resulting saturation curve at first rises more rapidly than a rectangular hyperbola, i.e. the curve is antisigmoid. 2. If the ouabain-sensitive K influx or the ouabain-sensitive Cs influx is measured in Na-free solutions at a fixed low concentration of K or Cs and at varying concentrations of Li, the influx decreases monotonically as the Li concentration rises and there is no evidence of competitive activation. 3. These findings can be accounted for by a model which proposes that there are two binding sites for K or Cs and that both the singly loaded and doubly loaded pump is capable of transport. 4. Extracellular Na changes the shape of both the K and the Cs saturation curve from antisigmoid to sigmoid. Dixon plots (1/ouabain-sensitive influx versus Na concentration at fixed K or Cs concentration) are linear at intermediate concentrations of K or Cs. 5. Na does not change the rate of K influx if the measurements are made at nearly saturating K concentrations using cells with nearly saturating internal Na concentrations. The effect of outside Na cannot therefore be explained by any mechanism which requires that Na alter the Vmax of the pump. 6. Measurement of the ouabain-sensitive Cs influx as a function of the external Cs concentration in solutions with different fixed Na concentrations results in curves which change from antisigmoid in Na-free solutions to sigmoid as the Na concentration rises. Dixon plots are linear at all but the lowest and highest Cs concentrations. 7. The resulting curves are best fit by equations which result from a model which proposes that Na acts both as a dead-end competitive inhibitor and as a heterotropic allosteric effector. Simpler models which propose either that Na acts solely as a dead-end competitive inhibitor or as a heterotropic allosteric effector do not fit as well as the more complicated model. 8. The combined competitive inhibition and allosteric effector model also describes adequately the relation between the ouabain-sensitive K influx and external K concentration measured at different external Na concentrations.

Binding Sites↗
PubMed1977

Kinetics of the inhibition of the Na-K pump by tetrapropylammonium chloride.

1. The tetrapropylammonium ion (TPA) acts as a mixed-type (with K) inhibitor of the Na-K pump. The kinetics of the process suggest that combination of the pump with a single TPA ion is sufficient for inhibition. 2. TPA inhibits the partial reactions of the Na-K pump (the uncoupled Na outflux, the Na-Na exchange, and K-K exchange). 3. TPA inhibits ouabain binding to the pump and this inhibitory effect is enhanced by external Na. The inhibitory effect of TPA on the pump rate is also promoted by external Na. 4. A Lineweaver-Burk plot of the reciprocal of the ouabain-sensitive K influx versus the reciprocal of the external K concentration is approximately a straight line if the measurements are made in Na-free solutions. TPA increases the apparent Michaelis constant (K 1/2) for K and the plot remains straight. 5. The Lineweaver-Burk plot is parabolic when the measurements are made in solutions which contain Na. TPA both increases the apparent K 1/2 for K and makes the curve more parabolic. 6. The characteristics of pump inhibition by TPA are similar to those for strophanthidin. In both cases the kinetic behaviour is consistent with a model in which the inhibitor binds: with greatest affinity to the pump form free of K; with less affinity to the pump form with a single bound K; and with least affinity to the pump form with two bound K.

Ammonium Chloride↗
PubMed1977

Kinetic evaluation of the Na-K pump reaction mechanism.

1. The ouabain-sensitive K influx was measured at varying external K concentrations ([K]o) and at several fixed internal Na concentrations ([Na]c). The cells were nominally K-free and the solutions Na-free. Both the apparent maximal velocity (VM) and the apparent Michaelis constant for K (KK) increased as Nac increased. The ratio app. VM/app. KK increased with increasing Nac. 2. The ouabain-sensitive Cs influx was measured at varying external Cs concentrations and at several fixed Nac in K-free cells and Na-free solutions. Both app. VM and app. Kcs increased as Nac increased and the ratio app. VM/app. Kcs increased with increasing Nac. 3. The data were evaluated in terms of ping-pong model and a simultaneous model for the pump reaction mechanism. The simultaneous model described the data adequately and the ping-pong models did not. 4. The K influx was measured at varying external K concentrations in solutions containing Na and at a low and high Nac; the cells contained K. The relation between the pump rate and the external K concentration was sigmoid. A Hill equation was fitted to the data. KK was higher in the high Nac cells, but the Hill coefficient (n) was not altered as Nac increased. 5. The K influx was measured at varying internal Na concentrations and two fixed external K concentrations; the cells contained K. The relation between the pump rate and Nac was sigmoid. When a Hill equation was fitted to the data, it was found that KNac was higher at the high external K concentration, but n was the same at both K concentrations.

Biological Transport, Active↗
PubMed1974

Interaction of external K, Na, and cardioactive steroids with the Na-K pump of the human red blood cell.

The interaction of extracellular Na (Na(o)), K (K(o)), 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 Na(o), K(o), and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K(1/2) of the pump for K(o), but does not change the form of the relation between the reciprocal of the active K influx ((i)M(K) (P-1)) and the reciprocal of [K(o)] ([K(o)](-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 (i)M(K) (P-1) vs. [K(o)](-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 Na(o). 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 Na(o) 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 K(o) than with strophanthidin.

Cardanolides↗
PubMed1974

Antibody-induced alterations in the kinetic characteristics of the Na:K pump in goat red blood cells.

The kinetic characteristics of the Na:K pump in high potassium (HK) and low potassium (LK) goat red cells were investigated after altering the intracellular cation concentrations. At low concentrations of intracellular K (K(c)), increasing K(c) at first stimulates the active K influx in HK cells, but at higher K(c) the pump is inhibited. These results suggest that in HK cells K(c) acts both at a stimulatory site at the inner aspect of the pump and by competition with intracellular Na (Na(c)) at the Na translocation sites. In LK cells, K(c) inhibits the active K influx and the sensitivity of LK cells to inhibition is much greater than the sensitivity of HK cells. Exposure of LK cells to an antibody (anti-L), raised in an HK sheep by injection of LK sheep cells, increased the active K influx at any given K(c). The effect of the antibody was greater at higher intracellular K concentrations, and in cells with very low concentrations of K the antibody had little effect on the pump rate. The failure of anti-L to stimulate the pump in low K(c) LK cells was not due to failure of the antibody to bind to the cells. Anti-L combining at the outer surface of the cell reduces the affinity of the pump at the inner surface for K at the inhibitory sites. The maximal pump rate in LK cells at optimal Na and K concentrations is less than the maximal pump rate of HK cells under the same circumstances.

Animals↗
PubMed1974

Interaction of HK and LK goat red blood cells with ouabain.

The characteristics of the interaction of Na-K pumps of high potassium (HK) and low potassium (LK) goat red blood cells with ouabain have been determined. The rate of inhibition by ouabain of the pump of HK cells is greater than the rate of inhibition of the pumps of LK cells. Treatment of LK cells with an antibody (anti-L) raised in HK sheep by injecting LK sheep red cells increases the rate of inhibition of the LK pumps by ouabain to that characteristic of HK pumps; reduction of intracellular K (K(c)) in LK cells increases the rate at which ouabain inhibits their pumps and exposure of these low K(c) cells to anti-L does not affect the rate of inhibition. There is considerable heterogeneity in the pumps of both HK and LK cells in the rate at which they interact with ouabain or the rate at which they pump or both. LK pumps which are sensitive to stimulation by anti-L bind ouabain less rapidly than the remainder of the LK pumps and exposure to antibody increases the rate at which ouabain binds to the sensitive pumps; the difference between the two types of pumps disappears if intracellular K is very low. The calculated number of ouabain molecules bound at 100% inhibition of the pump is about the same for HK and LK cells. Although exposure to anti-L increases the apparent number of ouabain binding sites in LK cells at normal K(c), it does not alter the apparent number of sites in LK cells when K(c) has been reduced.

Animals↗
PubMed1972

Recoupling the Na-K pump.

Human red blood cells display under appropriate circumstances a ouabain-sensitive K-K exchange when the flux measurements are made using radioisotopes. Such an exchange complicates measurements of the coupling of Na outflux to K influx in cells which are partially depleted of energy sources by deprivation of glucose since the K-K exchange has been found to be increased in depleted cells. When the measurements of flux are made by estimating net cation movements chemically, it is found that glucose deprivation results in a fall in both ouabain-sensitive Na outflux and ouabain-sensitive K influx. Since both fluxes fell in concert, there is no reason for believing that the fluxes are not coupled or that the source of ATP for the Na outflux is different from that for the K influx.

Adenosine Triphosphate↗