The coupling of downhill ion movements associated with reversal of the sodium pump in human red cells

1. Previous work on the incorporation of inorganic phosphate (P(i)) into ATP has suggested reversal of the chemical reactions of the Na pump in human red cells. A study has now been made of the associated movements of Na and K.2. The efflux of K, and the influx and efflux of Na were measured. When the Ringer was without K, the loss of cell K was inhibited by ouabain, and the ouabain-sensitive component of K efflux (0.36 mu-equiv.ml.(-1).hr(-1)) required external Na.3. The exchange of Na was also inhibited by ouabain. When influx and efflux of Na were measured simultaneously in K-free Ringer there was an excess of ouabain-sensitive influx over efflux of about 0.36 mu-equiv.ml.(-1).hr(-1). This difference balanced the ouabain-sensitive K efflux, and was not found with 10 mM-external K. The Na and K movements appear to be coupled and to be mediated by reversal of the Na pump.4. The net uptake of Na sensitive to ouabain was 0.38 mu-equiv.ml.(-1).hr(-1) for red cells incubated in K-free Ringer, and the net loss of K under the same conditions was 0.58 mu-equiv.ml.(-1).hr(-1) in rough keeping with the unidirectional flux values.5. Oligomycin decreased Na influx and efflux to the same extent as ouabain.6. There appears to be a coupled downhill movement of Na and K that is abolished both by inhibitors of the Na pump and by external K which promotes normal transport of Na outwards and K inwards.     

Potassium: potassium exchange catalysed by the sodium pump in human red cells

1. When red cells were so depleted of Na that Na:K exchange had almost ceased, the ouabain-sensitive K efflux seen in K-containing media was accompanied by an almost equal ouabain-sensitive K influx.2. This suggests that the Na pump in these cells was carrying out a one-for-one K:K exchange across the erythrocyte membrane.3. 30-40% of the (42)K efflux from resealed ghosts was sensitive to ouabain when the ghosts contained 1 mM-ATP, 2 mM orthophosphate, 10 mM-K and less than 1 mM-Na, and the suspending medium contained 10 mM-K and 0-Na, choline being the predominant cation.4. In resealed ghosts, the rate of K:K exchange saturated as internal K was increased, and was half-maximal at about 10 mM-K.5. When internal ATP was maintained with a phosphocreatine:creatine phosphokinase regenerating system, K:K exchange saturated as internal ATP was increased, and was half-maximal at about 100 muM-ATP.6. The rate of K:K exchange did not depend on whether the ADP concentration was roughly the same as the ATP concentration or very much less, suggesting that ADP did not affect the rate of K:K exchange.7. GTP, ITP and UTP were unable to substitute for ATP in supporting K:K exchange. CTP was a poor substitute.8. There was no evidence to support the hypothesis that K:K exchange is accompanied by a ouabain-sensitive hydrolysis of ATP.9. Internal Na was a strong inhibitor of ouabain-sensitive K efflux from ghosts containing 9 mM-K. 4 mM-Na was sufficient to produce 90% inhibition.10. The rate of K:K exchange depended on the orthophosphate concentration inside the ghosts (confirming Glynn, Lew & Lüthi, 1970). The curve obtained suggested that the rate was half-maximal at about 1.7 mM orthophosphate.11. These experiments suggested that inhibition by internal K is an important factor affecting the Na efflux from intact red cells. Experiments measuring Na:K exchange as a function of internal Na in low-K ghosts supported this hypothesis.12. The significance of these findings is discussed.     

Thallium and the sodium pump in human red cells

1. Thallium (Tl) inhibits the ouabain-sensitive K influx in human red cells in high-Na medium. At 1 mM external K concentration [K(o)], the ouabain-sensitive K influx decreases steadily with increasing Tl concentration, up to 0.9 mM outside; at 0.17 mM-K(o), however, Tl stimulates the ouabain-sensitive K influx below 0.1 mM-Tl(o) and inhibits it at higher concentrations.2. In a K-free medium in which all except 5 mM-Na is replaced by choline, and into which red cells show zero control ouabain-sensitive Na efflux, Tl is able to support ouabain-sensitive Na efflux up to 2.1 m-mole/l. cells.hr following a sigmoid activation curve which is half-maximal between 0.03 and 0.05 mM-Tl(o) and that follows two-site kinetics up to 0.1 mM-Tl(o). Beyond 0.15 mM-Tl(o), the Tl-activated ouabain-sensitive Na efflux attained is inhibited slightly.3. When the ouabain-sensitive Na efflux is measured at 5 mM-Na(o) and 5 mM-K(o), increasing concentrations of Tl have little effect on it, 0.9 mM-Tl(o) inhibiting by some 14%; in similar conditions, the ouabain-sensitive K influx is inhibited by about 40%.4. The dependence of ouabain-sensitive K influx on external K concentration at 5 mM-Na(o), which follows a slightly sigmoid curve in the absence of Tl, changes to hyperbolic at 0.06 mM-Tl(o) at the same time that ouabain-sensitive K influx is inhibited. The fitted V(max) values for ouabain-sensitive K influx are the same in the presence and in the absence of 0.06 mM-Tl(o).5. In high-Na cells, loaded by nystatin treatment, the ouabain-sensitive K influx measured at 0.2 mM-Na(o) follows a hyperbolic curve between 0.05 and 0.4 mM-K(o), and is inhibited by Tl in a strictly competitive fashion.6. The effects of Tl on ouabain-sensitive Na efflux and ouabain-sensitive K influx are interpreted in terms of a high-affinity substitution for K at the external K sites of the Na pump and suggest that in human red cells Tl can be actively transported inwards in exchange for internal Na.7. Thallium can inhibit about 25% of the ouabain-insensitive Na efflux into 5 mM-Na(o) and part of this inhibition occurs with a high Tl-affinity; the ouabain-insensitive K influx is inhibited by Tl both in high-Na and in 5 mM-Na medium, but with a different concentration dependence than the ouabain-insensitive Na efflux.     

Sodium, potassium, water, and haemoglobin in the packed red cells of severe thalassaemia

Sodium, potassium, water, and the mean corpuscular haemoglobin concentration were determined in the packed erythrocytes of children with severe thalassaemia. The concentration of sodium in the packed red cells was higher than normal in a significant proportion of children with thalassaemia whereas potassium in the packed cells and sodium and potassium in the plasma were normal.     

Potassium activated phosphatase from human red blood cells. The mechanism of potassium activation

1. The kinetic behaviour of the K-activated phosphatase in human red blood cell membranes has been investigated. The concentration of Mg required to give optimal activation is independent of substrate and K concentration, suggesting that Mg combines with the enzyme at a site that is independent of and non-interacting with the substrate and K sites.2. The effects of K are competitively antagonized by Na. Ouabain in suitable concentrations selectively abolishes the activating effect of K.3. Comparison between the hydrolysis of acetylphosphate by intact red cells and by fragmented ghosts suggests that the active site for the substrate is only accessible at the internal surface of the cell membrane.4. The plot of the total rate of p-nitrophenylphosphate hydrolysis versus substrate concentration can be fitted at any K concentration by a rectangular hyperbola. The effect on the total rate of increasing K concentration is exerted mainly on the apparent affinity for the substrate, which increases about 5 times as K goes from 0 to 55 mM. There is also a much smaller increase in the maximum velocity (about 1.3 times) for the same range of K concentrations.5. If the difference between the activity in the absence and in the presence of K is plotted as a function of substrate concentration, the curves obtained are no longer hyperbolic but pass through a maximum and then tend to a lower value.6. This kinetic behaviour can be explained much more easily by assuming that a single enzyme is responsible for the hydrolysis of the substrate in the presence and in the absence of K. A simple kinetic model based on this assumption was developed and when experimentally determined constants were fitted into the equations that predict its behaviour a reasonably good agreement between theory and experiment was obtained.7. In the ;single enzyme' model inhibitors that selectively abolish the K-dependent activity would act by blocking the combination of the enzyme with K. A simple treatment based on this idea was developed for the case of Na and its predictions were fulfilled by the experimental results.8. In the ;single enzyme' model the K-coupled hydrolysis is always larger than the difference between the rates in the presence and in the absence of K, and when K concentration is non-limiting the K-coupled rate is equal to the total rate.     

The interaction of adenosinetriphosphate and inorganic phosphate with the sodium pump in red cells.

1. An increase in the intracellular concentration of inorganic phosphate (Pi) reduces the rate of the Na:K exchange catalysed by the Na pump in red cells. The inhibitory effect of Pi is exerted on the maximum rate of flux, Pi having no appreciable effect on the apparent affinity of the Na pump for either internal Na or external K. The effect of Pi is exerted along a rectangular hyperbola which tends to zero as Pi tends to infinity and is half-maximal at about 17 mM internal Pi. 2. Pi does not modify the rate of Na:Na exchange catalysed by the Na pump. 3. A reduction in the intracellular concentration of ATP reduces the maximum rate of Na:K exchange having no effect on the apparent affinity for either internal Na or external K. 4. The effects of ATP and Pi are mutually independent. 5 The lack of effect of ATP and Pi on the apparent affinity for internal Na is compatible with the idea that the affinity of the inner sites of the Na pump remains constant during a pump cycle. 6. The lack of effect of ATP on the apparent affinity for external K and the independence between the effects of ATP and Pi are difficult to explain if the only effect of ATP were its combination at a phosphorylating site. 7. The apparent affinities for K and phosphate become independent of the concentration of ATP, if it is assumed that in our experimental range the phosphorylating site is fully saturated with ATP, the rate of pumping being controlled by the state of occupation of a second non-phosphorylating site whose affinity for ATP is much lower. 8. The lack of effect of Pi on the apparent affinity for external K seems to indicate that during Na:K exchange the conformations of the pump that predominate are endowed with a reactivity towards inorganic phosphate and have the same high affinity for K in both their phospho and their dephospho states. 9. The kinetic behaviour of the Na pump in regard to its interactions with inner and outer cations, ATP and Pi seems to indicate that, in contrast with what happens with soluble allosteric proteins, in the active transport system ligand-induced changes in the reactivity are more important than ligand-induced changes in affinity. In this respect therefore the Na pump behaves as an allosteric 'V system'.     

Effect of extracellular potassium on the loss of potassium from human red blood cells treated with propranolol

The Ca⁺⁺-dependent propranolol-induced increase of K⁺ permeability of human red blood cells, well documented in previous studies, was found to depend on extracellular K⁺. This was shown by studying the passive transport of ⁸⁶Rb and the loss of bulk cellular K⁺ in both K⁺-free and K⁺-containing media. The maximal effect of propranolol was achieved with 5–10 mM K⁺ in incubation media. The external K⁺ could be substituted with TI⁺, but not with Na⁺. When added after propranolol, extracellular K⁺ failed to initiate the effect of propranolol on membrane permeability. The cell/medium distribution of permeant ²⁰⁴TI showed that the propranolol-induced increase of K⁺ permeability did not result in considerable hyperpolarization of the red blood cell membrane. The data obtained seem to be more consistent with a counter-transport model for explaining the propranolol effect than with a mechanism based on free diffusion of K⁺ through the membrane.     

Ion movements in human red cells independent of the sodium pump

1. A study was made of the dependence on external Na of the movements of Na and K in human red cells. Special attention was given to ouabain-insensitive movements. The effect of internal Na on Na influx, and the influence of some sulphydryl inhibitors on ion movements and metabolism was also investigated.2. External Na stimulated ouabain-insensitive Na efflux and K influx. There was also a ouabain-insensitive component of Na influx that was raised on increasing the internal Na concentration. Exchange diffusion of Na appears to occur in the presence of ouabain and external K.3. Net transport of Na and K in the presence of ouabain was independent of external Na, as was also lactate production.4. Ethacrynic acid partially inhibited the Na pump; the Na-dependent components of Na efflux and K influx in the presence of ouabain were completely inhibited by ethacrynic acid. Both ouabain-sensitive and ouabain-insensitive adenosinetriphosphatase activities were inhibited by ethacrynic acid indicating a non-specific effect of this compound. Iodoacetamide decreased only the ouabain-insensitive ATPase activity.5. The results suggest that when the Na pump is blocked by ouabain, part of the residual ion movements can be attributed to exchange diffusion.     

Sodium and potassium permeability of red blood cells in dependence of the pH

Summary In rabbit red cells, it has been found that the permeability to sodium and potassium ions depends on the hydrogen ion concentration.The sodium and potassium net flux showed maxima at distinctly different pH-values: sodium at pH 6.6, and potassium at pH 7.0.Studies with radioisotopes indicated that the pH-dependent maxima of thenet fluxes were due to an increase of thedownhill movement of sodium and potassium ions. The uphill movement, in contrast to the downhill movement, was little influenced by the hydrogen ion concentration.The downhill movement as well as the uphill movement haveQ ₁₀-values of more than 2.0.The potassium influxmeasured under the influence of ouabain is in close agreement to the influxcalculated by means of the efflux rates and the concentration gradients.In order to explain the changes in permeability, a hypothesis has been developed assuming that structural elements of the membrane with ampholytic properties such as proteins and phosphatides are responsible for the change in permeability caused by variations in the hydrogen ion concentration.

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Zusammenfassung Die Durchlässigkeit von Kaninchenerythrocyten für Natrium-bzw. Kaliumionen ist in hohem Maße von der Wasserstoffionenkonzentration abhängig.Sowohl der Natrium- als auch der Kalium-Nettoflux zeigen ein Maximum, aber bei unterschiedlichen pH-Werten: Natrium bei pH 6,6 und Kalium bei pH 7,0.Untersuchungen mit Radioisotopen ergaben, daß die für den Nettoflux gefundenen Maxima vorwiegend auf einer Beeinflussung der Bergabbewegung beruhen. Die Abhängigkeit der Bergaufbewegung von der Wasserstoffionenkonzentration ist im Gegensatz zur Bergabbewegung gering.Sowohl die Bergauf- als auch die Bergabbewegung von Kalium und Natrium sind stark temperaturabhängig und haben einenQ ₁₀-Wert über 2,0.Der unter Ouabaingemessene Kaliuminflux stimmt sowohl hinsichtlich der pH-Abhängigkeit als auch des Ausmaßes mit dem aus den Effluxraten und den Konzentrationsgradientenerrechneten Kaliuminflux gut überein.Die Diskussion der Befunde geht von der Vorstellung aus, daß Strukturelemente der Erythrocytenmembran mit ampholytischem Charakter, wie Proteine und Phosphatide, für die durch Wasserstoffionen hervorgerufenen Änderungen des Natrium- bzw. Kaliumfluxes verantwortlich sind.

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A report based on these results was given at the joint meeting of the Swiss and German Societies of Biochemists in Oct. 1960, Zürich, Switzerland.     

Bim regulates BCR-induced entry of B cells into the cell cycle

BH3-only Bcl-2 homologs are key regulators of the intrinsic apoptotic pathway. In particular, Bim, is critical for mediating apoptosis of hematopoietic cells including B cells. While studies using Bcl-2 Tg mice have defined an important role for Bcl-2 in cell cycle control, the role of BH3-only proteins is less clear. Using Bim KO mice, we show that Bim is required for B cells to enter the cell cycle normally. Bim KO B cells had reduced cell division compared to WT B cells in response to BCR, TLR3 or TLR4 signaling, whereas Bim deficiency did not affect TLR9-induced B cell division. Cell cycle progression in BCR- and LPS-stimulated Bim KO B cells was blocked at the G0-G1 stage. BCR-induced p130 degradation and pRb hyperphosphorylation on Ser807/811, which are critical for G1 entry, were reduced in Bim KO compared to WT B cells. Likewise, BCR-induced p27(Kip1) degradation was decreased in Bim KO compared to WT B cells. These defects in BCR-induced cell cycle entry correlated with a proximal defect in BCR-mediated intracellular calcium release in Bim KO B cells. Our results suggest that the balance of pro- and anti-apoptotic Bcl-2 family proteins is critical for controlling both cell cycle progression and apoptosis in B cells.     

Studies on the mechanism of entry of vaccinia virus in animal cells

Summary In order to study the mechanism of entry of vaccinia virus into cells the fate of virion associated polypeptides was investigated during infection of african green monkey kidney (BSC-40) cells with 35 S-methionine labelled virus. Approximately 12–15 percent of the virion polypeptides were degraded to acid-soluble products by 3 hours post-infection. Proteolysis was inhibited (50 percent) by methylamine, suggesting a lysosomal site of degradation. Neither methylamine or chloroquine inhibited virus infectivity or uncoating indicating a non-acid endocytic mechanism of entry. Subcellular fractionation studies on density gradients indicated that the bulk of the input virion polypeptides were associated with the plasma membrane fraction. In addition, input virion DNA was partially resolved from the membrane fraction. The results are most consistent with a mechanism of entry involving fusion of the virus with the plasma membrane.With 7 Figures     

病原菌入侵宿主细胞的机制研究进展

传染病病原菌是对人类和动物健康的最大威胁。深入研究病原菌如何入侵宿主细胞的机制是揭示传染病的关键。经过长期的进化革兰氏阴性菌发展形成几种分泌途径破坏宿主细胞,包括普通分泌途径、Ⅰ型、Ⅱ型、Ⅲ型和Ⅳ型分泌途径。这些病原菌主要是通过摧毁宿主细胞的细胞骨架、调节信号转导途径和破坏宿主细胞的免疫机制达到感染宿主细胞的目的。本文首先就病原菌毒力因子的分泌途径进行了初步介绍,然后对目前有关病原菌如何入侵宿主细胞以及入侵宿主细胞后如何破坏细胞的结构和功能进行了概述。