Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Effects and mechanisms of action of ionophorous antibiotics valinomycin and salinomycin-Na on Babesia gibsoni in vitro

Valinomycin and salinomycin-Na, 2 ionophorous antibiotics, exhibited in vitro antibabesial activities against Babesia gibsoni that infected normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes, which completely lack Na,K-ATPase activity. The level of parasitemia of B. gibsoni was significantly decreased when the parasites were incubated in culture medium containing either 10(-1) ng/ml valinomycin or 10(2) ng/ml salinomycin-Na for 24 hr. Four-hour incubation in the culture medium containing 5 μg/ml salinomycin-Na led to the destruction of most parasites. In contrast, when the parasites infected canine erythrocytes containing high potassium (HK) and low sodium concentrations, i.e., HK erythrocytes, the in vitro antibabesial activities of both ionophorous antibiotics seemed to be weakened, apparently due to the protection by the host cells. Therefore, differential influences of ionophorous antibiotics on LK and HK erythrocytes were observed. In LK erythrocytes, the intracellular concentrations of potassium, sodium, and adenosine triphosphate (ATP) were not modified, and hemolysis was not observed after incubation in the medium containing each ionophorous antibiotic. These results suggested that these ionophorous antibiotics did not affect cells without Na,K-ATPase, and directly affected B. gibsoni. In HK erythrocytes, the ionophorous antibiotics increased the intracellular sodium concentration, and decreased the intracellular potassium and ATP concentrations, causing obvious hemolysis. Additionally, the decrease of the intracellular ATP concentration and the hemolysis in HK erythrocytes caused by valinomycin disappeared when the activity of Na,K-ATPase was inhibited by ouabain. These results indicate that modification of the intracellular cation concentrations by the ionophorous antibiotics led to the activation of Na,K-ATPase and increased consumption of intracellular ATP, and that the depletion of intracellular ATP resulted in hemolysis in HK erythrocytes. Moreover, the antibabesial activity of valinomycin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations. This showed that the intracellular cation concentration in the parasites was not modified in those media and would remain the same.     

Inhibition of Na,K-ATPase activity reduces Babesia gibsoni infection of canine erythrocytes with inherited high K, low Na concentrations

Babesia gibsoni multiplies well in canine red blood cells (RBCs) containing high concentrations of potassium (HK), reduced glutathione, and free amino acids as a result of an inherited high Na,K-ATPase activity, i.e., HK RBCs. To determine the role of Na,K-ATPase in the multiplication of B. gibsoni, the effect of ouabain on the proliferation of the parasites in HK RBCs was investigated. To determine the direct effect of ouabain on the parasites, the proliferation of the parasites in normal canine RBCs containing low potassium (LK) and high sodium concentrations, i.e., LK RBCs, which completely lack Na,K-ATPase activity, was observed. Ouabain at 0.1 mM significantly suppressed the multiplication of B. gibsoni in HK RBCs in vitro, whereas it had no effect on the parasites in LK RBCs. The results suggest that the multiplication of B. gibsoni in HK RBCs depends mainly on the presence of Na,K-ATPase in the cells. Therefore, the effects of ouabain on the intracellular cation and free amino acid composition of the HK RBCs were examined. In HK RBCs incubated with ouabain, a marked decrease in the concentration of potassium and an increase in sodium were observed, together with a decrease in the number of parasitized cells. These results suggest that the intracellular cation composition maintained by Na,K-ATPase might be advantageous to the parasites. Moreover, the concentrations of some free amino acids, i.e., asparagine, aspartate, glutamate, glutamine, glycine, and histidine, were markedly decreased in HK RBCs incubated with ouabain. Decreased concentrations of the free amino acids induced by inhibition of Na,K-ATPase seemed to affect the multiplication of B. gibsoni in HK RBCs. Based on these results, it is clear that the high Na,K-ATPase activity in HK RBCs contributes to the proliferation of B. gibsoni by maintaining high potassium and low sodium concentrations, as well as high concentrations of some free amino acids in the cells.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells.

Binding of highly purified 125I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75-100 and 500-750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3-6 x 10(3) in HK (MM) and 1.5-4 x 10(3) in LK (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2-1.8 x 10(3) in LL and less in LM(LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na+K+ pumps measured previously in these cells by 3H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na+K+ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and /or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics, which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na+K+ pump molecules is more complex than that in goat red cells previously reported by others.     

Regulation of Cell Volume by Active Cation Transport in High and Low Potassium Sheep Red Cells

A model cell which controls its cation composition and volume by the action of a K-Na exchange pump and leaks for both ions working in parallel is presented. Equations are formulated which describe the behavior of this model in terms of three membrane parameters. From these equations and the steady state concentrations of Na, K, and Cl, values for these parameters in high potassium (HK) and low potassium (LK) sheep red cells are calculated. Kinetic experiments designed to measure the membrane parameters directly in the two types of sheep red cells are also reported. The values of the parameters obtained in these experiments agreed well with those calculated from the steady state concentrations of ions and the theoretical equations. It is concluded that both HK and LK sheep red cells control their cation composition and volume in a manner consistent with the model cell. Both have a cation pump which exchanges one sodium ion from inside the cell with one potassium ion from outside the cell but the pump is working approximately four times faster in the HK cell. The characteristics of the cation leak in the two cell types are also very different since the HK cells are relatively more leaky to sodium as compared with potassium than is the case in the LK cells. Both cell types show appreciable sodium exchange diffusion but this process is more rapid in the LK than in the HK cells.     

Active sodium and potassium transport in high potassium and low potassium sheep red cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K_1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K_1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.     

L-antigen and active potassium transport in HK and LK red cells of Barbary sheep (Ammotragus lervia)

1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 4. Active K+ transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K+ transport with anti-L. One HK animal (2BA2) showed some stimulation of active K+ transport, and also absorbed some anti-L from antisera, suggesting that Lp antigen is present on these red cells. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.     

Enzymatic modification of the L and M antigens in LK and HK sheep erythrocytes and their membranes

Summary This paper reports on the effect of two hydrolytic enzymes, neuraminidase and trypsin, on the interaction of blood group L-positive low-potassium-type (LK) and blood group M-positive high-potassium-type (HK) sheep red cells with their respective isoimmune antisera. It was found that treatment of LK and HK red cells with neuraminidase did not change the interaction of these cells with their homologous antibodies as measured by K⁺-pump flux, complement-mediated immune hemolysis and absorption of antibody. Similarly, trypsin pretreatment of LK and HK red cells did not interfere with the hemolytic action of anti-L and anti-M antibodies, respectively. In striking contrast, however, it was observed that pretreatment of LK cells with trypsin rendered these cells insensitive to the K⁺-pump stimulating antibody present in the anti-L serum.     

Preferential hemolysis of postnatal calf red cells induced by internal alkanlinization

Red blood cells from neonatal calves, but not from adult cows, rapidly hemolyze in buffered 300 mM solutions of a variety of nonelectrolytes and amino acids. Of these compounds, sucrose is chosen to elucidate the mechanism by which this preferential hemolysis takes place. As in other mammalian red cells, both calf and cow cells are found to be impermeable to sucrose and, in an isosmolar sucrose solution, to undergo volume shrinkage caused by the net loss of chloride ions with concomitant increase in intracellular pH. To test the potential role of intracellular pH change associated with chloride loss in promoting hemolysis, intracellular pH was altered by: (a) a direct addition of fixed acid or base to sucrose solution; (b) the removal of dissolved CO(2) from sucrose solution; and (c) the addition of cells to isotonic NaHCO(3) solution in the absence of sucrose. In all cases, only calf and not cow cells underwent hemolysis. Moreover, 4-acetamido-4’-isothiocyano-2,2’-stilbene disulfonic acid, a potent anion transport inhibitor, completely protected calf cells from hemolysis and caused a nearly total inhibition of both chloride loss and intracellular alkalinization. Furthermore, the hemolytic process is closely related to the integrity of a membrane protein, the band 3 protein, which can be cleaved to varying degrees by the combined treatment of pronase and lipase. Hemolysis is progressively inhibited as the band 3 protein undergoes proteolysis, until a total inhibition of hemolysis takes place when almost all of the band 3 protein is digested into smaller protein components with a mol wt of 65,000 and 35,000 daltons. These results suggest that the intracellular alkalinization process leading to a structural instability of the membrane band 3 protein is responsible for this calf cell hemolysis.     

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.     

(Na,K)-ATPase and Ouabain binding in reticulocytes from dogs with high K and low K erythrocytes and their changes during maturation

The present study demonstrated that dog reticulocytes had considerable amounts of (Na,K)-ATPase, but lost it rapidly during maturation into erythrocytes. Furthermore, reticulocytes from dogs possessing erythrocytes characterized with high (Na,K)-ATPase activity and high K, low Na concentrations (HK dogs; Maede, Y., Inaba, M., and Taniguchi, N. (1983) Blood 61,493-499) had more ouabain binding sites than cells from normal dogs (LK dogs). Our results were as follows: i) The maximal binding capacities (Bmax) for ouabain binding at equilibrium were approximately 0 and 1,500 binding sites/cell in LK and HK dog erythrocytes, respectively. ii) Reticulocytes from LK dogs possess approximately 5,700 ouabain binding sites/cell. iii) The Bmax value for ouabain in HK reticulocytes was about 10,000 sites/cell, being 2-fold that in LK reticulocytes. iv) Ouabain-sensitive fluxes of 24Na and 42K in each type of reticulocyte were compatible with the number of ouabain binding sites on the cells. v) Ouabain binding capacity, as well as (Na,K)-ATPase activity, in the reticulocytes from LK dogs fell rapidly to nearly zero during the maturation into erythrocytes. vi) Although reticulocytes from HK dogs also showed a similar regression of (Na,K)-ATPase during maturation, they retained a certain number of ouabain binding sites even after maturation, resulting in the high activity of (Na,K)-ATPase in HK erythrocyte membrane.     

Transitory postnatal hemolysis of calf red cells by amino acids

Summary Among the amino acids which can be solubilized to give a concentration of 300 mM at near physiological pH, histidine and proline caused a complete hemolysis of newborn calf but not of adult cow red cells within 20 to 30 minutes at 38°C. While hydroxyproline, valine, and serine resulted in a partial lysis of calf cells, threonine, glutamine, and glycine were inefective. In this communication, emphasis has been focused on the mode of the lytic process by histidine, which was found to be affected by several governing parameters including the pH, temperature and the extracellular salts in the solution. Unlike human red cells suspended in isotonic histidine, both calf and cow cells lost little Na and K ions. In the presence of 300 mM histidine, both calf and cow cells displayed an instantaneous uptake of histidine amounting to 20 to 45 moles/ml RBC followed by a slow influx rate of 0.25 to 0.5 moles/ml RBC×min. The extent to which histidine entry was allowed by the cell was counterbalanced by Cl^– efflux, resulting in little change in cell volume prior to hemolysis. Moreover, histidine-induced hemolysis can by prevented by 1 mM or lower PCMBS without at discernible effect on histidine influx suggesting a possible membrane lesion or damage at the outer surface of the cell.Hemolysis induced by histidine decreased substantially when a calf reached two months of age which time the red cells containing the fetal hemoglobin are virtually depleted. The results of hemoglobin electroiphoresis obtained during this postnatal period revealed that those cells resistant to histidine hemolysis almost invariably contain the adult type hemoglobin suggesting a selective, speicific action of the amin acid on the featal cells.A preliminary report of these data has been presented at the 19th Annual Meeting (1975) of the Biophysical Society, Philadelphia, Pennsylvania.     

The Effect of Valinomycin on Potassium and Sodium Permeability of HK and LK Sheep Red Cells

A cyclic depsipeptide antibiotic, valinomycin, was found to produce increased selective permeability of the plasma membranes of HK and LK sheep red blood cells to potassium but not to sodium ions. The compound had relatively little effect on the active extrusion of sodium from HK sheep red blood cells or on the Na + K-stimulated ATPase activity of membranes derived from these cells. It is proposed that the selective cation permeability produced by this compound depends primarily on steric factors, particularly the relationship between the diameter of the ring and the effective diameter of the ion. The significance of these results for the problem of the mechanism of ionic selectivity in natural membranes is discussed.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

The number of Na:K pump sites on red blood cells from HK and LK lambs

Lambs of known genotype with respect to the locus determining cation composition of red cells were obtained by selective matings. Numbers of K⁺ pump sites per cell were determined on HK and LK lambs 10–20 days postnatal by simultaneously determining [³H]ouabain binding and inhibition of active K⁺ transport. Red cells from HK lambs were indistinguishable from adult HK cells with regard to the K⁺ pump flux and number of pump sites. Cells from genetically LK lambs had pump fluxes and numbers of pump sites intermediate between those from adult HK and LK sheep. The results suggest that the change in cation composition and in the K⁺ pump during the first 60 days in genetically LK lambs can be correlated with a reduced number of K⁺ pump sites.     

The HK/LK polymorphism of erythrocyte cation content in two wild species of east African bovids: demonstrated in wildebeest but not in African buffalo

Concentrations of K and Na were determined in erythrocytes from wildebeest and African buffalo resident in Tanzania. The object was to determine if these species possess the HK/LK polymorphism which is typical of other bovid species, but not of other mammals. The polymorphism is characterized by individuals that are either of the HK phenotype (high K and low Na concentrations in red cells) or LK phenotype (low K and high Na concentrations in red cells). Wildebeest were shown to be polymorphic, with the distribution of phenotypes resembling that in domestic cattle. By contrast the polymorphism was not found in the African buffalo; all 59 individuals examined exhibited the HK phenotype. This was unexpected, since the polymorphism has been observed in five divergent bovid species, including two species (water buffalo and domestic cow) that are closely related to the African buffalo, and classified in the same subfamily (Bovinae). The most parsimonious interpretation of this pattern is that the trait was lost from the African buffalo after species in the Bovinae diverged. The biological significance of the HK/LK polymorphism, and reasons for its presence or absence among species in the Bovidae, remain obscure.     

The effect of sodium periodate treatment on the modulation of the sodium pump in low-potassium type (LK) sheep red cells by the L antigen

1. The action of sodium periodate and neuraminidase on active and passive K+ transport in low-potassium type (LK) sheep red cells was investigated in relation to the contribution of the Lp and Ll antigens. 2. Active K+ transport in LK sheep red cells was not affected by treatment with sodium periodate (2 mM), or with neuraminidase. 3. Passive K+ transport in LK sheep red cells was increased by sodium periodate treatment in a concentration-dependent manner. The increase was not Cl- dependent, and so differed from the increased passive K+ uptake resulting from N-ethylmaleimide treatment. 4. HK sheep red cells treated with sodium periodate showed small increases in passive K+ uptake, and N-ethylmaleimide treatment used sequentially with sodium periodate resulted in further small increases in passive K+ uptake. 5. In LK sheep red cells the stimulation of active K+ transport by anti-L was impaired by 50% in cells treated with sodium periodate (2 mM) and was slightly lowered in cells treated with neuraminidase. 6. In LK sheep red cells inhibition of passive K+ transport by anti-L was not impaired by sodium periodate treatment (2 mM), or by neuraminidase treatment.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.