The activation of the sodium pump in pig red blood cells by internal and external cations.

A study has been made with pig red blood cells of the activation of the sodium pump by internal and external cations. Cell Na and K concentrations were altered using a PCMBS cation loading procedure. The procedure was characterised for resultant ionic conditions, maintenance of ATP levels and fragility. The activation of the sodium pump by external K was measured in cells suspended in choline (Na-free) solutions. External Cs was used as a substitute for K and elicited lower rates of pump activity. Both the Vmax and apparent Km for 42K influx and 134Cs influx increased as internal Na concentration was raised (within the non-saturating range). Vmax/apparent Km ratios for cation influx were constant. Raising external Cs concentration exerted a similar influence on pump activation by internal Na: both the maximum pump velocity and the apparent Na-site dissociation constant (K'Na) increased. The results provide evidence for a transmembrane connection between cation binding sites on opposite faces of the membrane and are consistent with a consecutive model for the sodium pump in pig red blood cells.

The uptake and hydrolysis of p-nitrophenyl phosphate by red cells in relation to ATP hydrolysis by the sodium pump.

1. The hydrolysis of p-nitrophenyl phosphate has been studied in human red cells, ghosts and haemoglobin-free membranes to see whether hydrolysis was related to the functioning of the sodium pump.2. The cell membrane restricted p-nitrophenyl phosphate entry into cells and was rate-limiting for hydrolysis by the large amount of intracellular soluble phosphatase. The uptake was inversely proportional to the external chloride concentration, and inhibitors (phloretin and persantin) of inorganic phosphate uptake also reduced p-nitrophenyl phosphate uptake and hydrolysis. The entry mechanism of p-nitrophenyl phosphate appears to be similar to that of inorganic phosphate.3. p-Nitrophenyl phosphate was hydrolysed in cells almost entirely by ouabain-insensitive phosphatases, both soluble and membrane bound. In ghosts containing less soluble enzyme than cells there was still no component of p-nitrophenyl phosphate hydrolysis that was related to the sodium pump in being sensitive to external potassium or ouabain.4. Haemoglobin-free membranes lacking soluble p-nitrophenyl phosphatase required potassium for optimum p-nitrophenyl phosphate hydrolysis and this part was inhibited by ouabain as was also ATP hydrolysis by the sodium pump. The rates of potassium-dependent and potassium-independent hydrolysis were each increased about threefold on decreasing the electrolyte concentration from 150 to 25 mM. The response was found whether the main electrolyte was potassium chloride, sodium chloride, choline chloride or Tris chloride. In contrast, the changes were not found when the osmotic pressure was varied to the same extent with non-electrolytes. p-Nitrophenyl phosphate hydrolysis was thus activated on lowering the ionic strength. The soluble enzyme was similarly affected and the effect was reversible.5. The membrane ATPase activity was unaffected by the changes in ionic strength which markedly altered p-nitrophenyl phosphate hydrolysis.6. These results with red cell membranes show that the ionic strength under physiological conditions largely prevents the hydrolysis of p-nitrophenyl phosphate, but not of ATP. A possible mechanism is discussed in terms of the effect of different structures of water in electrolyte and non-electrolyte solutions on the enzymic activity of the sodium pump.

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.

The influence of the chloride gradient across red cell membranes on sodium and potassium movements.

1. A study has been made to see whether active and passive movements of sodium and potassium in human red blood cells are influenced by changing the chloride gradient and hence the potential difference across the cell membrane.2. Chloride distribution was measured between red cells and isotonic solutions with a range of concentrations of chloride and non-penetrating anions (EDTA, citrate, gluconate). The cell chloride concentration was greater than that outside with low external chloride, suggesting that the sign of the membrane potential was reversed. The chloride ratio (internal/external) was approximately equal to the inverse of the hydrogen ion ratio at normal and low external chloride, and inversely proportional to external pH. These results show that chloride is passively distributed, making it valid to calculate the membrane potential from the chloride ratio.3. Ouabain-sensitive (pump) potassium influx and sodium efflux were decreased by not more than 20 and 40% respectively on reversing the chloride gradient, corresponding to a change in membrane potential from -9 to +30 mV. In contrast, passive (ouabain-insensitive) movements were reversibly altered - potassium influx was decreased about 60% and potassium efflux was increased some tenfold. Sodium influx was unaffected by the nature of the anion and depended only on the external sodium concentration, whereas ouabain-insensitive sodium efflux was increased about threefold. When external sodium was replaced by potassium there was a decrease in ouabain-insensitive sodium efflux with normal chloride, but an increase in low-chloride medium.4. Net movements of sodium and potassium were roughly in accord with the unidirectional fluxes.5. The results suggest that reversing the chloride gradient and, therefore, the sign of the membrane potential, had little effect on the sodium pump, but caused a marked increase in passive outward movements of both sodium and potassium ions.

The metabolic response of brain slices to agents affecting the sodium pump.

1. Slices of brain cortex from rabbits were incubated in Ringer solution and in Ringer modified by the removal of calcium and sodium, and the addition of ouabain, oligomycin or extra potassium. The potassium content of the tissue, the oxygen consumption and the lactate production from glucose were measured and found to be interrelated.2. Incubation in high-K Ringer caused an increase in oxygen consumption that was prevented by ouabain, oligomycin and deprivation of sodium. Lactate production was also raised, but this increase was unaffected by ouabain and raised further by oligomycin.3. Calcium omission raised metabolism; the tissue K content was unaffected. Oligomycin always decreased oxygen consumption and raised lactate production further. The metabolic responses to calcium, potassium, ouabain and oligomycin depended on sodium.4. After anaerobic incubation, the tissue potassium concentration was still 5 times higher than that in Ringer. It was unaffected by oligomycin but lowered markedly by ouabain.5. The synergistic effects of sodium with potassium, oligomycin, calcium, and calcium plus ouabain suggest that the metabolic responses of brain cortex slices to a high-K Ringer depend on the operation of the sodium pump.

Reconstitution of the sodium pump from protein and phosphatidylserine: features of ouabain binding.

1. A study has been made of ATP splitting and ouabain binding to the sodium pump reconstituted from protein and phosphatidylserine.2. Ouabain was bound to protein alone, but when phosphatidylserine was added, binding was increased threefold. The stimulation resembled the course of activation of sodium-dependent ATPase activity.3. EGTA partly simulated the activation of ATPase by phosphatidylserine but did not enhance binding.4. The dissociation constant for the enzyme-ouabain complex was 3.5 x 10(-8)M. The turnover number (2,000 molecules of ATP per minute) and the number of receptor sites (3.8 x 10(13) per mg protein) were calculated.5. The results provide further evidence of the involvement of phosphatidylserine in the action of the sodium pump.

The mode of inhibition by calcium of cell-membrane adenosine-triphosphatase activity.

1. The mechanism of the inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase by calcium was investigated with an enzyme preparation from rabbit kidney cortex and with membranes of human erythrocytes. 2. CaATP, rather than ionic Ca(2+), acts as a competitive inhibitor, competing with MgATP in the Na(+)-plus-K(+)-activated adenosine-triphosphatase reaction. 3. There appears to be no competition between calcium and Na(+) for the activation of adenosine triphosphatase. 4. The inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase of cell membranes by low concentrations of CaATP and the consequent need of intact cells to keep the cytoplasmic concentration of calcium low relative to that of magnesium suggests a raison d'être for the mitochondrial calcium pump.

The involvement of phosphatidylserine in adenosine triphosphatase activity of the sodium pump.

1. A study has been made to see whether the ATPase of the Na pump is activated by phospholipids.2. Solubilization of a membrane-containing (microsomal) fraction from ox brain cortex with deoxycholate removed most or all of the ouabain-inhibited ATPase and p-nitrophenylphosphatase (pNPPase) activities. Addition of sonicated dispersions of crude commercial samples of phosphatidic acid, phosphatidylinositol or phosphatidylserine stimulated both enzymic activities, but neither phosphatidylcholine nor phosphatidylethanolamine affected them. After partial purification by chromatography, however, definite activation was demonstrated only with that component of crude samples which migrated like phosphatidylserine.3. The ATPase activity dependent on phosphatidylserine was eliminated by removal of Na and K or by addition of ouabain and was substantially inhibited by oligomycin. The corresponding component of pNPPase activity was partially inhibited by ouabain or by removal of most of the K, but was little affected by oligomycin. The phosphatidylserine-dependent enzyme(s) therefore exhibited properties characteristic of the ATPase of the Na pump.4. Phosphatidylserine dispersions similarly activated both the ATPase and the pNPPase of the particulate enzyme preparations, untreated with deoxycholate.5. The results suggest that the system for active transport of Na and K involves a complex unit of phosphatidylserine and the protein of the ATPase.

The temperature dependence of activation by phosphatidylserine of the sodium pump adenosine triphosphatase.

1. Treatment of rabbit brain homogenates with deoxycholate reduced ouabain-insensitive ATPase sixfold and subsequently adding phosphatidylserine had no effect. Ouabain-sensitive ATPase was made entirely latent but it was fully restored on adding phosphatidylserine.2. Temperature and pH were varied to see if the reconstituted system resembled that in the original membranes. Linear Arrhenius plots were always obtained with the homogenate, and the activation energy was higher for the ouabain-sensitive than for the ouabain-resistant enzyme.3. A break at about 15 degrees C was found in the Arrhenius plot of the reconstituted enzyme, but there was no break without added phosphatidylserine or when ouabain was added. The break suggests that the conformation and catalytic activity of the enzyme protein depended on the physical state of phosphatidylserine.

The connexion between the ion-binding sites of the sodium pump.

1. A study has been made of K influx into human red blood cells in order to determine the influence of internal Na on the affinity of the Na pump for external K. Cells were prepared to contain minimal K and two Na concentrations (about 10 and 30 mueqiv/ml. cells) and incubated in solutions with a range of K concentrations. 2. In choline (Na-free) Ringer, activation of K influx by external K was hyperbolic. The Km for external K increased as the internal Na concentration was raised. The increase was greater than the increase in K influx with saturating external K. The Km for external K increased towards a limiting value as the internal Na concentration was raised. 3. In contrast, in Na-Ringer, the activation by external K was sigmoidal and the affinity for external K was independent of the internal Na concentration. 4. K influx was measured at two submaximal levels of external K with a range of internal Na. The affinity for internal Na fell as external K was raised. 5. The results suggest that in the absence of competition between Na and K on the same side of the membrane, there is a connexion between the ion-binding sites of the Na pump.

Some aspects of adenosine triphosphate synthesis from adenine and adenosine in human red blood cells.

1. The synthesis of ATP has been studied in human erythrocytes. Fresh cells showed no net synthesis of ATP when incubated with adenine or adenosine, although labelled adenine was incorporated into ATP in small amounts.2. Cold-stored cells (3-6 weeks old) became progressively depleted of adenine nucleotides but incubation with adenosine or adenine plus inosine restored the ATP concentration to normal within 4 hr. Incorporation of labelled adenine or adenosine into the ATP of incubated stored cells corresponded to net ATP synthesis by these cells.3. Synthesis of ATP from adenosine plus adenine together was 75% derived from adenine and only 25% from adenosine, indicating that nucleotide synthesis from adenine inhibits the simultaneous synthesis of nucleotide from adenosine.

The control by internal calcium of membrane permeability to sodium and potassium.

1. A study has been made of the relationship between the concentration of internal calcium and the permeability of human red cell membranes to sodium and potassium.2. Fresh red cells contain very little calcium, but after being depleted of ATP by ageing they took up calcium from Ringer solution. The entry was unaffected by external sodium and potassium but was markedly pH dependent. When supplied with energy, calcium-loaded cells actively extruded calcium by a saturable process which was also unaffected by the distribution of sodium and potassium across the membranes. The activity of the calcium pump was sufficient to maintain the low internal concentration found under physiological conditions.3. Raising internal calcium in metabolically poor cells caused a loss of cell potassium which was greater than the concomitant sodium gain. These changes were reversed when ATP was supplied. External calcium had no effect. The increase in permeability to sodium and potassium was enhanced by the simultaneous addition of fluoride, and, even more so, of iodoacetate. These inhibitors had no effect on membrane permeability unless calcium was also present. Inosine potentiated the action of fluoride and iodoacetate in causing potassium loss, by allowing more calcium to enter the cells.4. The results suggest that the permeability of human red cell membranes to sodium and potassium is regulated by internal calcium, which in turn is controlled by a calcium pump that utilizes ATP.

The influence of external sodium ions on the sodium pump in erythrocytes.

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.

The influence of ions on the labelling of adenosine triphosphate in red cell ghosts.

1. The ionic composition of human red cell ghosts and suspending Ringer solutions have been varied independently. Measurements were made of the incorporation of [(32)P]o-phosphate ((32)P(i)) into ATP associated with different concentration gradients of Na and K across the membrane.2. Some incorporation of (32)P(i) was always found irrespective of the ionic composition of ghosts or media. However, additional labelling of energy-rich phosphate occurred when low Na, high K ghosts were incubated in a high Na, K-free medium. This did not occur when there was only a gradient of either Na or K. Downhill movements of both Na into and K out of the ghosts were needed for the extra labelling.3. Even in the presence of suitable ionic gradients, the extra incorporation was prevented by ouabain or by adding a small amount of external K sufficient to facilitate normal operation of the Na pump.4. Increase in internal P(i) stimulated the incorporation.5. The results show that the conditions for forward and backward running of the ATPase system associated with the Na pump are such that both reactions cannot proceed optimally at the same time.

Potassium transport and nucleoside metabolism in human red cells.

1. A study has been made of active cation transport in relation to the metabolism of nucleosides and deoxynucleosides in human red blood cells.2. Lactate production from inosine depended on the cellular inorganic phosphate available for phosphorolysis and was raised to some 4 times the normal rate from glucose. Inosine inhibited glucose metabolism according to the amount of glucose-6-phosphate produced. Guanosine behaved similarly, but glucose-6-phosphate was not formed from adenosine, deoxyinosine and deoxyguanosine.3. Glucose metabolism was related to the ATP content of the cells, which was raised by incubation with adenosine, or with inosine plus adenine.4. Adenosine and deoxyadenosine raised the potassium influx above the values found with inosine and deoxyinosine under conditions when the lactate production was constant. Active potassium influx was correlated with the ATP concentration in the cells, but independent of the lactate production when the latter was raised above normal.