Na-K Transport Research Articles

CHARACTERISTICS OF ACTIVE NA-K TRANSPORT IN ERYTHROCYTES IN CHILDREN WITH UREMIA

Purpose of the study: To evaluate the active Na-K transport in the following aspects of uremia: degree of azotemia, effect of therapeutic measures, relation to growth and hypertension.Methods: Erythrocyte Na-K-ATP-ase by measuring the capacity of hemoglobine free membranes to generate phosphate from ATP. Erythrocyte Na+, K+ and ATP were determined.Material: 15 children in different stages of uremia aged/months to 16 years.Results: All children had Na-K-ATP-ase activities within the range for corresponding ages. Crossectionally no correlation was seen between the levels of Na-K-ATP-ase and serum creatinine. Longitudinally a decrease of Na-K-ATP-ase and increase of erythrocyte Na-K ratio were seen. Dietary treatment did not change the levels of Na-K-ATP-ase, erythrocyte Na-K ratio and ATP. A single hemodialysis for 3-5 hours slightly increased Na-K-ATP-ase and erythrocyte Na-K ratio and repeated hemodialyses further increased the Na-K-ATP-ase. Renal transplantation markedly increased Na-K-ATP-ase and decreased erythrocyte Na-K ratio. A distinct feature of hypertensive uremics was a low erythrocyte Na-K ratio combined with a high Na-K-ATP-ase.Conclusion: The most striking effect upon parameters indicating active Na-K transport was seen after renal transplantation. The increased extracellular volume seen in hypertensive uremics could be explained by increased active Na-K transport.

SODIUM CONTENT OP RED BLOOD CELLS IN ACUTE RENAL FAILURE AND HYPERTENSION

The erythrocyte is a valuable model for the study of the metabolism of elektrolytes. Sodium metabolism of red blood cells (rbc) is altered in hypertension and renal diseases. Therefore we investigate the intracellular electrolytes in rbc of 23 children with acute renal failure. 12 children suffered from HUS, 11 from dehydration, septicemia etc. 10 of them had elevated blood pressure. Na+ was measured by flame photometry.In the majority the sodium content of rbc was elevated: Na+ 10.7 ± 3.5 mmol/1 in HUS, 10.2 ± 5.5 mmol/1 in others ( normal values: 6.1 ± 0.6 mmol/1). In hypertensive patients Na ± was significant elevated: Na+ 13.6 ± 6.1 mmol/1, 8.3 ± 2.2 mmol/1 ( p < 0.02 ).Retention of sodium and disturbances of Na-K transport by uremic toxins induce slight elevation of sodium in acute renal failure. The reasons of the higher concentration of Na+ in rbc are yet speculative. Intracellular sodium elevation is one of several factors producing hypertension. The clinical course was frequently complicated in children with high sodium content of rbc.

The apparent discrepancy of ouabain inhibition of cation transport and of lymphocyte proliferation is explained by time-dependency of ouabain binding.

Mitogenesis of human blood lymphocytes in culture is inhibited by concentrations of ouabain that are approximately one order of magnitude lower than those that block Na and K transport. For example, the 50% inhibition (ID50) of Na-K transport, 280 nM, is seven-fold greater than the Id50 for RNA synthesis, DNA synthesis, or blastogenesis, approximately 40 nM. Yet, inhibition of transport and consequent reduction in cell K is considered responsible for the effects of ouabain on mitogenesis. Since synthetic processes are assessed at least 24 hours after lymphocyte stimulation, this discrepancy could be explained by either 1) a progressive increase in K leak, or 2) a progressive inhibition of Na-K transport by ouabain during 24 hours of PHA treatment. We found that the lymphocyte membrane leak rate of K increased immediately after PHA treatment but did not increase further from 4 to 24 hours. In contrast, the ouabain sensitivity of 42K uptake was markedly increased with time: ID50 for 42K uptake of 35 nM at 24 hours as compared to 280 nM at 30 minutes. Measurement of ouabain binding revealed a seven-fold increase in the lymphocyte-associated ouabain after 24 hours compared to binding at 1 hour. These data indicate that the dose response of ouabain inhibition of active K transport and lymphocyte proliferation are closely correlated if one considers the slow membrane binding of ouabain at low concentrations.

Effects of epinephrine, ouabain, and insulin in experimental autoimmune myasthenia gravis.

To gain a clue to the target of anti-AChR antibody, rats with acute and chronic experimental autoimmune myasthenia gravis (EAMG) that was induced by immunization with Narke anti-acetylcholine receptor (AChR) were studied using agents acting on active Na-k transport. Postsynaptic response to epinephrine was defective in chronic EAMG with high titers of antibody, suggesting that active Na-K transport system modulated by cyclic adenosine monophosphate (AMP) may be affected primarily by antibody. Sensitivity to ouabain was less than normal in acute EAMG and became close to normal when treated with anticomplementary factor. Findings suggest that acute EAMG is a case of functional denervation. Normal response to insulin occurred in all phases of EAMG.

Thyroid hormones and the energetics of active sodium-potassium transport in mammalian skeletal muscles.

1. The steady-state heat production rate (E) of soleus muscles obtained from adult mice in various thyroid states was measured in a perfused microcalorimeter. The ouabain-suppressible fractions of E and 42K influx were compared and the energetic efficiency of active Na-K transport assessed. 2. Hypothyroidism with plasma thyroxine concentrations below 1 microgram/100 ml. was induced by pretreatment with 131I or perchlorate. In soleus muscles isolated from treated animals, mean E values were 25.1 +/- 0.7 and 24.2 +/- 0.5 mcal.g wet wt.-1.min-1 for the 131I and the perchlorate series respectively, i.e. about 30% lower than the control level (36.3 +/- 1.5 mcal.g wet wt.-1.min-1). Following triiodothyronine treatment, E was increased by about 45%. 3. In muscles from hypothyroid (131I and perchlorate series), euthyroid and hyperthyroid mice ouabain (10(-3) M) induced a rapid decrease in E of 1.6 +/- 0.1 and 1.4 +/- 0.1, 2.5 +/- 0.2, and 4.3 +/- 0.6 mcal.g wet wt.-1.min-1 respectively, i.e. between 6 and 8% of E. 4. In muscles obtained from hypothyroid, euthyroid and hyperthyroid mice, the ouabain-suppressible component of 42K influx was 0.17 +/- 0.04, 0.31 +/- 0.02 and 0.45 +/- 0.02 micromole. g wet wt.-1.min-1 respectively. Whereas the total number of ouabain binding sites varied appreciably with the thyroid status, the Na-K contents of soleus or diaphragm muscles showed no significant changes. 5. Notwithstanding the parallelism between the changes in basal E and ouabain-sensitive components of E and K influx with the thyroid status, it is concluded that active NA-K transport cannot be considered a primary effector of thyroid thermogenesis in intact mammalian skeletal muscle. The direct contribution of active NA-K transport to this thermogenesis was indeed small compared with the over-all cellular energy dissipation. 6. The minimum over-all energetic efficiency of the transport process in the intact muscles (30--35%) was not dependent on the thyroid status.

Properties of Na‐K pump in primary cultures of kidney cells

Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period. The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange. Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemide-sensitive K:K exchange).

Does cytoplasmic alkalinization trigger mitochondrial energy dissipation in the brown adipocyte?

Indirect calorimetry measurements showed that brown fat thermogenesis was very sensitive to modifications of intra-cellular pH induced by extracellular acid-base perturbations. Specific blockage of active Na-K transport by ouabain inhibited the thermogenic response only in acidosis and more efficiently when the glycoside was administered before the catecholamine stimulus than when it was added after the full calorigenic response had developed. It is suggested that the catecholamine stimulus might initiate a positive feed-back alkalinization of the cytoplasm, concomitant with activation of Na-K transport.

Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells.

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of greater than 1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.09--1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0--3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the spleen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol-rich red cells is decreased in the presence of the spleen. Although active Na-K transport is not influenced by cholesterol enrichment of human red cells, several carrier-mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol. Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of red cells in vivo.

The effect of catecholamines on Na—K transport and membrane potential in rat soleus muscle

1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (E(M)) has been investigated in soleus muscles isolated from fed rats.2. In a substrate-free Krebs-Ringer bicarbonate buffer adrenaline (ADR) (6 x 10(-6)M) increased (22)Na efflux by 83%, (42)K influx by 34%, and E(M) by 10%. Similar effects were exerted by noradrenaline (NA), phenylephrine, salbutamol and isoprenaline. The effects of ADR on Na-K transport and E(M) were suppressed by ouabain (10(-3)M) and propranolol (10(-5)M), but not by thymoxamine (10(-5)M) or tetracaine (10(-4)M).3. Following 90 min of incubation in the presence of ADR (6 x 10(-6)M), the intracellular K/Na-ratio was increased threefold. NA produced almost the same change, and both catecholamines seem to induce a new steady-state distribution of Na and K which can be maintained for several hours in vitro.4. The effect of ADR on (22)Na efflux and E(M) could be detected at concentrations down to 6 x 10(-9) and 6 x 10(-10)M, respectively, and half-maximum increase was obtained at around 2 x 10(-8)M. NA was at least one order of magnitude less potent.5. The effect of low concentrations of ADR on (22)Na efflux was potentiated by theophylline (2 mM). When added together, dibutyryl-cyclic AMP and theophylline mimicked the action of ADR on (22)Na efflux, (42)K influx, Na/K content and E(M). Ouabain (10(-3)M) also suppressed the effect of dibutyryl-cyclic AMP and theophylline on Na-K transport.6. Following the addition of ouabain (10(-3)M), E(M) rapidly dropped from a mean of -71 to -63 mV, and then showed a slow linear fall for up to 4hr.7. The hyperpolarization induced by ADR was associated with a decrease in membrane conductance, (22)Na influx and (42)K efflux. The time course and the response to ouabain suggests that all of these effects are secondary to stimulation of the active coupled transport of Na and K.8. It is concluded that in rat soleus muscle, the active Na-K transport is electrogenic and susceptible to stimulation by catecholamines via beta-adrenoceptors. This effect is mediated by adenyl cyclase activation and may account for the increase in E(M) and the intracellular K/Na ratio.

Active Na-K transport and the rate of ouabain binding. The effect of insulin and other stimuli on skeletal muscle and adipocytes.

1. The effect of stimulation or inhibition of active Na-K transport on [(3)H]ouabain binding has been investigated in isolated soleus muscles and adipocytes.2. In rat soleus muscle, the ouabain-sensitive component of (42)K influx was stimulated by insulin (100 m-u/ml.), adrenaline (6 x 10(-6)M), and by pre-incubation with veratrine (10(-5)M) or in a K-free buffer. In all of these instances, the rate of ouabain binding was increased by 41-113%. Conversely, pre-treatment with tetracaine (0.2 mM) decreased the (42)K-influx and diminished the rate of [(3)H]ouabain binding by 36%.3. Neither insulin, adrenaline or tetracaine produced any detectable change in the total number of ouabain-binding sites (as measured under equilibrium conditions) in rat soleus muscle.4. In mouse and guinea-pig soleus muscle and in fat cells isolated from rats, insulin also increased the rate of [(3)H]ouabain binding without producing any significant change in the total number of ouabain-binding sites.5. Both in soleus muscle and the epididymal fat pad of the rat, there was a linear correlation between (42)K influx and the initial rate of [(3)H]ouabain binding.6. It is concluded that the rate of ouabain binding is determined significantly by the rate of active Na-K transport, but within the time intervals studied (4-6 hr) stimulation or inhibition of the Na pump does not lead to any appreciable change in the total number of Na pumps. It seems unlikely that the stimulation of active Na-K transport by insulin or adrenaline is due to unmasking or de novo synthesis of Na pumps.

Thyroid calorigenesis in isolated, perfused rat liver: minor role of active sodium-potassium transport.

1. The effects of ouabain on hepatic oxygen uptake, cell membrane potential, and Na-K transport were examined at 37 degrees C during non-recirculating perfusion of isolated livers from fasted normal rats and rats treated with triiodothyronine (T3). The perfusate was Krebs-Ringer bicarbonate buffer containing albumin and bovine erythrocytes. 2. Treatment with T3 increased the rate of hepatic oxygen uptake by 30% (i.e. by 0-83 (micromole/min) per gram liver). 3. After shifting to perfusate containing 2-5 mM ouabain, a 4-5 mV depolarization and maximal rates of net hepatic K release and Na uptake occurred within 2 min in both thyroid states. These changes were not accompanied by any significant change in the rates of hepatic oxygen uptake. 4. T3-treatment increased the maximal, post-ouabain net flux of K by 29% (i.e. by 0-52 (muequiv/min) per gram liver). The T3-indlced increase in the net flux of Na (19%) did not achieve statistical significance. 5. In either thyroid state, the observed passive fluxes of Na and K were calculated to be balanced by active vluxes at the expense of 5-6% of the observed rate of hepatic oxygen uptake. 6. The results indicate that hyperthyroidism may enhance the rate of hepatic Na-K transport, but the energy expenditure due to this process appears to be too small to make any important contribution to thyroid calorigenesis in perfused rat liver.

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions.

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.

Microcalorimetric determination of energy expenditure due to active sodium-potassium transport in the soleus muscle and brown adipose tissue of the rat.

1. The resting heat production rate (E) of soleus muscles from young rats and brown adipose tissue from adult rats was measured by means of a perfusable heat flux microcalorimeter in the absence and presence of ouabain. In the soleus muscle, the acute response of E to ouabain was compared with the ouabain-suppressible components of 22Na-efflux and 42K-influx. 2. In standard Krebs-Ringer bicarbonate buffer, ouabain (10(-3)M) induced an immediate but transient decrease in E of around 5%. Both in muscle and adipose tissue this was followed by a progressive rise in heat production rate. 3. When the medium was enriched with Mg (10 mM), ouabain produced a sustained decrease in E of the same magnitude as in the standard medium and the secondary rise was less marked or abolished. Under these conditions, in the soleus muscle, ouabain inhibited E by 5% (i.e. by 1-76 +/- 0-22 mcal.g wet wt.-1.min-1), 22Na-efflux by 58% (0-187 +/- 0-013 micronmole. g wet wt.-1.min-1) and 42K-influx by 34% (0-132 +/- 0-028 micronmole. g wet wt.-1.min-1). 4. When the muscles were loaded with Na by pre-incubation in K-free Mg-enriched medium, the addition of K (3mM) induced an immediate ouabain-suppressible increase in E of 2-98 +/- 0-33 mcal. g wet wt.-1.min-1 and a concomitant stimulation of 22Na-efflux of 0-388 +/- 0-136 micronmole. g wet wt.-1.min-1. 5. Maximum Na/ATP ratios for the active Na-K transport process were computed, with no assumption as to the in vivo free energy of ATP hydrolysis. These were 2-1, 1-9 and 2-3 under the conditions described in paragraphs (2), (3) and (4) respectively. 6. The calculated reversible thermodynamic work associated with active Na-K transport corresponded to 34% of the measured ouabain-induced decrease in E. On the premise that the maximum efficiency of the cellular energy conservation processes is 65%, this estimate indicates that the minimum energetic efficiency of ATP utilization by the active Na-K transport process in mammalian muscle is 52%.

Aerobic and anaerobic metabolism in smooth muscle cells of taenia coli in relation to active ion transport.

1. The O2 consumption and lactic acid production of the guinea-pig's taenia coli have been studied in relation to the active Na-K transport, in order to estimate the ratio: active Na extrusion/active K uptake/ATP hydrolysis. 2. By applying different procedures of partial metabolic ingibition, it was found that a reactivation of the active Na-K transport in K-depleted tissues could occur in an anaerobic medium, provided glucose was present and in an aerobic medium free of added metabolizable substrate. The active Na-K transport was rapidly blocked in an anaerobic-substrate free medium. 3. Readmission of K to K-depleted tissues under aerobic conditions stimulates both O2 consumption and lactic acid production. While the O2 consumption creeps up slowly and requires 50 min to reach control values, the aerobic lactic acid production increases to a maximum within 10 min and decreases again during the next 50 min to its steady-state value. 4. A reactivation of the Na-pump in K-depleted cells in a N2-glucose medium causes an immediate increase of the lactic acid production, which decreases to its control value after 60 min. The maximal increase in anaerobic lactic acid production during reactivation of the Na-K pump is a function of [K]O. The system can be cescribed with first order kinetics having a Vmax = 0-72 mumole.g-1 f. wt. min-1 and a Km = 1-1 mM. 5. By varying the glucose concentration of [K]O during reactivation of the Na-K pump, different Na-K pumping rates can be obtained. The ratios net Na extrusion/ATP or net K accumulation/ATP amount to -1-32 +/- 0-19 (36) and 1-02 +/- 0-11 (36), in the experiments with different glucose concentrations. Taking into account the interference by net passive fluxes, one can estimate a ratio:active Na transport/active K transport/ATP, of 1-7/0-8/1. This ratio is not very different from the values observed in other tissues.

Extracellular space and ionic distribution in rat ventricle.

Extracellular space and ionic distribution in rat ventricle.

Na-K transport in rat liver slices in hemorrhagic shock.

Na-K transport in rat liver slices in hemorrhagic shock.

Temperature adaptation of active sodium-potassium transport and of passive permeability in erythrocytes of ground squirrels.

Unidirectional active and passive fluxes of (42)K and (24)Na were measured in red blood cells of ground squirrels (hibernators) and guinea pigs (nonhibernators). As temperature is lowered, "active" (ouabain-sensitive) K influx and Na efflux were more greatly diminished in guinea pig cells than in those of ground squirrels. The fraction of total K influx which is ouabain sensitive in red blood cells of ground squirrels was virtually constant at all temperatures, whereas it decreased abruptly in guinea pig cells as temperature was lowered. All the passive fluxes (i.e., Na influx, K efflux, and ouabain-insensitive K influx and Na efflux) decreased logarithmically with decrease in temperature in both species, but in ground squirrels the temperature dependence (Q(10) 2.5-3.0) was greater than in guinea pig (Q(10) 1.6-1.9). Thus, red blood cells of ground squirrel are able to resist loss of K and gain of Na at low temperature both because of relatively greater Na-K transport (than in cells of nonhibernators) and because of reduced passive leakage of ions.

Membrane potential of brown adipose tissue. A suggested mechanism for the regulation of thermogenesis.

Membrane potentials were recorded in isolated segments of interscapular brown adipose tissue from rats. After equilibration at 29 degrees C in Krebs-Ringer bicarbonate buffer a mean value of -51 +/- 4 mv (SD) was found. This level could be maintained for up to 5 hr. The mean effective membrane resistance was 1.35 +/- 0.45 megohm. The membrane potential was a function of the extracellular potassium concentration. Ouabain (10(-6)-10(-3)M) and incubation in K-free buffer produced progressive depolarization. Epinephrine and norepinephrine in concentrations as low as 10(-8) g/ml produced a prompt depolarization. Cooling of the tissue and lowering of the oxygen tension caused a marked and reversible decrease in the membrane potential. In tissue obtained from cold-adapted rats, the membrane potential was considerably diminished. 6Assuming that the membrane potential is some function of the Na permeability of the plasma membrane it is suggested that an increase in the rate of active Na-K transport and ensuing ADP formation might contribute to the increase in respiration seen during exposure to thermogenic stimuli.

Stability of the potassium content of cerebrospinal fluid and brain.

Stability of the potassium content of cerebrospinal fluid and brain.

The role of membrane phosphoglycerate kinase in the control of glycolytic rate by active cation transport in human red blood cells.

When the internal Na of human red cells is raised, both K influx and lactate production increase and become more sensitive to the inhibitory action of ouabain. This occurs with either glucose or purine nucleoside as substrate. Fresh whole hemolysates enriched with Na and Mg will convert intermediates above the triose phosphate dehydrogenase step to lactate at a rate which is slowed by ouabain. Intermediates beyond the phosphoglycerate kinase step (PGK) are metabolized at a very rapid rate which is not affected by ouabain. No metabolic effects of ouabain were found in ghost-free hemolysates. Hemoglobin-free ghosts were shown to have both triose phosphate dehydrogenase and PGK activity. The rate of this two-enzyme sequence was found to be a function of the ADP concentration, being maximal when ADP > 0.35 mM. Initial addition of ATP to the ghost system rendered the forward rate of the sequence sensitive to the inhibitory action of ouabain. When the sequence was run in reverse, no inhibitory effect of ouabain could be demonstrated. It is concluded that membrane PGK is a point at which the Na-K transport system can influence the metabolic rate and that this action is possibly exerted via a compartmentalized form of ADP which is an immediate substrate for the ghost PGK.