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.     

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.     

The M-antigen in HK and LK sheep red cell membranes

Summary Red cells of all high-potassium-type (HK) sheep and of more than one half of all low-potassium-type (LK) sheep contained the M-antigen and were hemolyzed by iso-immune anti-M antiserum in presence of a guinea pig serum complement. It was characteristic for the hemolysis of HK red cells by the M-antiserum the all HK cells were ultimately hemolyzed at suboptimal antibody concentrations, provided the time of incubation at 37 °C was sufficiently long. Thus, the M-antigen appears to be expressed on all red cells of an individual HK sheep. The M-antibody was absorbed by HK red cells and their membranes with a high affinity, whereas M-negative LK red cells and their membranes did not bind the antibody. The ratio of the number of antibody units absorbed per cell or membrane to the number of antibody units required for lysis approached unity. The amount of antibody absorbed per membrane was unaffected by ouabain in the presence of ATP, Mg⁺⁺, Na⁺, and K⁺. The M-antigen activity depends on the integrity of the red cell membrane and was not detectable after lyophilization of HK membranes or in the membrane protein solubilized by n-butanol. The major M-antibody activity was found among the high molecular weight plasma proteins and may be attributed to the ₂ M globulins. Heterogeneity within the antibody fraction cannot be excluded since some hemolytic activity was detected in a chromatographic fraction containing predominantly -globulin. The relationship between the M-antigen and the Na⁺–K⁺ transport system in sheep red cell membranes is discussed.This work was presented in part at the 53rd annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J. 1969.     

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.     

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.     

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.     

The correlation of composition and morphology during the high to low potassium transition in single erythropoietic cells

The change from high potassium dog erythroid cells to low potassium red blood cells during erythropoiesis was investigated by X-ray microanalysis of single cells. A correlation of morphology and composition, using freeze-dried cryosectioned preparations, showed that during normal erythropoiesis in dog bone marrow the switch from high potassium to low potassium occurs during the change from early to late nucleated erythroid cells, and in synchrony with the beginning of iron accumulation. In contrast, during rapid erythropoiesis in dogs with phenylhydrazine-induced anemia, the most prominent change in cation composition as well as the accumulation of iron occurs during the reticulocyte stage in the peripheral blood. The determination of the absolute amounts of sodium and potassium per cell in stress reticulocytes of peripheral blood indicated that the changeover from high potassium to low potassium actually occurs by the loss of cellular potassium during volume reduction, with little change in the amount of cellular sodium. This suggests that maturation may involve a selective change in potassium permeability. Lastly, it was observed that not all cells followed the predominant pathway with respect to change in morphology, membrane permeability and hemoglobin synthesis. One particular subpopulation appeared to follow a sequence which expressed the complete HK to LK transition before the accumulation of any iron; this implies the possibility of completing protein synthesis in a low potassium intracellular milieu.     

Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: (1) HK membranes sonicated for 20 minutes, (2) HK membranes sonicated for 3 minutes, and (3) LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation (1) was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8 per cent of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations (2) and (3) but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation (2) was about 50 per cent that observed in HK membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation (1), I-ATPase activity in both preparations (2) and (3) was recovered in small but heavy (specific gravity > 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme(s) and large, light, phospholipid-containing components is discussed.     

LK sheep reticulocytosis: effect of anti-L on K influx and in vitro maturation

After massive hemorrhage, adult sheep with genotypically low potassium (LK) red cells temporarily produce high potassium (HK) cells with ouabain-sensitive K+ pump fluxes equivalent to mature HK red cells. In light of recent reports of different red cell volume populations accompanying the HK-LK transition also occurring in newborn LK sheep and the unresolved controversy over the effect of anti-L on K+ transport in these immature red cells, we have reexamined the K+ transport changes and the effect of anti-L in the newly formed HK cells at various times after anemic stress and under in vitro conditions. We found that approximately 7 d after bleeding, maximum reticulocytosis occurred in the peripheral blood. After separation by density centrifugation, the top 10% cell fraction contained 100% reticulocytes, with a mean cell volume 2.5 times larger than that of mature erythrocytes. These immature red cells were of HK type, and their K+ pump and leak fluxes were 30 and 10 times higher, respectively, than those found in mature LK cells. The new cells may possess HK- and LK- type pumps because K+ pump influx was significantly stimulated by anti- L. When separated by density centrifugation on days 9, 17, and 23 after bleeding, some of the cells apparently maintained their large size while gaining higher density. Large cells from day 9, kept in vitro for 22 h, showed anti-L-sensitive K+ pump and leak fluxes that declined within hours, paralleling the behavior of these cells in vivo, whereas cellular K+ levels changed much less. It is concluded that the newly formed red cells may belong to a stress-induced macrocytic cell population that does not acquire all of the characteristics of adult LK cells.     

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells [1]. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithio-threitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux [1]. Maximum ³H-NEM incorporation into the DTT-reduced thiols occurred at 50 M DTT. Saturation labelling by ³H-NEM of about 2 × 10⁴ diamide-protected thiols/cell occurred at 25 M NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier [2]. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.     

Interaction of HK and LK Goat Red Blood Cells with Ouabain

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

Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells.

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.     

The effect of anti-L on ouabain binding to sheep erythrocytes.

Binding of 3H-ouabain was studied in high potassium (HK) and low potassium (LK) sheep red cells. In particular, we investigated the effect of anti-L, an antibody raised in HK sheep against L-positive LK sheep red cells, on 3H-oubain binding and its relation to K+ -pump flux inhibition in LK cells. HK cells were found to have about twice as many 3H-ouabain binding sites and a higher association rate for 3H-ouabain than homozygous LL-type LK cells. The number of 3H-ouabain molecules bound to heterozygous LM-type LK cells is lower than that on LL cells, but the rate of ouabain binding is between that of HK and LL red cells. A close correlation was observed between the rates of 3H-oubain binding and fraction K+-pump inhibition. Exposure of LM and LL cells to anti-L did not affect the number of 3H-ouabain molecules bound at saturation, but increased the rates of glycoside binding and K+ -pump inhibition proportionately, so that for LK cells in the presence of anti-L, the rates of the two processes approximate those of HK cells. These data exclude the possibility that anti-L generates entirely new pump sites in LK sheep cells, but suggest that the antibody increases the affinity of the existing -a+ -K+ pumps for the glycoside.     

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.     

Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

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.     

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.     

Properties of a goat anti-L antibody: further evidence for heterogeneity of the L antigen.

The preparation and properties of an antibody (anti-L) against low potassium type (LK) goat red cells raised in a high potassium type (HK) goat are described. This reagent stimulated active potassium transport, but showed only weak serological activity against low potassium type (LK) sheep and goat red cells. The results are discussed in relation to the hypothesis that anti-L antibody has two specificities--a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L1y).     

Identification of a 25 kDa polypeptide associated with the L antigen in low potassium-type sheep red cells

Antisera to the L blood group antigen have been used, following radioiodination of low potassium-type sheep red cells and subsequent immunoprecipitation, to identify a polypeptide of the L antigen. Only LK, and not HK, cells express this 25 kDa component which is present in very low copy number.