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.     

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).     

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 anti-body has two specificities — a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L_ly.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

GENETIC VARIATION IN THE SHEEP RED BLOOD CELL

1. There are 7 well-established red-cell antigen (blood group) loci. The R-O system has 3 phenotypes, R, O and i, identified by the ‘naturally occurring’ antibodies, anti-R and anti-O. The R and O substances are also present in soluble form in some body secretions. The expression of R and O is controlled by a dominant gene I, epistatic in effect, at an independent locus from that of R. The systems, A, C, M-L, B, D and X-2 are identified by means of ‘immune-type’ antibodies, and several of the loci have multiple alleles. An isoenzymic form of serum alkaline phosphatase is associated with the R-O system. The frequency for the genes at the various loci has been determined in a limited number of breeds. 2. Some sheep red cells have high K+ and low Na+ concentrations (HK type, or Key), others have low K+ and high Na⁺ concentrations (LK type or Kea). Two other rare forms exist; Key type which is HK but with lower than normal K+ values, and Kep type which has approximately equal Na⁺ and K⁺ concentrations. The red cells of foetuses and newborn lambs have high K+ levels irrespective of their potassium genotype. HK cells have 3–4 times greater (Na+-K+)-activated ATPase activity, a 3–4 times increased rate of active K+ transport and a larger number of ouabain-binding sites than LK cells. Antigen M is present on homozygous HK and heterozygous LK red cells, and antigen L is present on homozygous and heterozygous LK red cells. Sensitization of LK cells with anti-L stimulates active K+ transport and ATPase activity and exposes a larger number of ouabain-binding sites in these cells. Anti-M has no effect. The red cells of newborn lambs only show weak L and M antigen activity. It is postulated that L antigen inhibits cation transport in LK cells by masking the pump sites on the membrane. Immature red cells in LK-type sheep have a high rate of active K+ transport and yet have L antigen present. No satisfactory explanation for this has yet been advanced. There is no conclusive evidence that the potassium types have any significance from the point of view of adaptation or sheep breeding. The potassium-gene frequencies are known for a large number of breeds. 3. Two allelic genes, Hb⁴ and Hb^B control 3 haemoglobin phenotypes, A, AB, and B. Foetal haemoglobin (HbF) is present in foetuses and newborn lambs. Sheep with HbA also synthesize small amounts of another haemoglobin (HbC) and under conditions of severe anaemia, synthesis of HbC takes over from that of HbA. No change in HbB is observed in anaemia. A rare haemoglobin (HbD) has been found in 3 Yugoslavian sheep. Hbs A, B, C and F differ in their physicochemical properties; they share the same alpha chains but their non-alpha chains differ in a number of amino acids. HbD differs from HbA in one amino acid in the alpha chain. Certain genetic aspects are discussed. There is some evidence that sheep with HbA are less fertile than those with HbB. The gene frequencies for Hb are known for a large number of breeds. 4. Two isoenzymic forms of carbonic anhydrase are found in red-cell lysates and these are controlled by a pair of allelic autosomal genes, producing 3 phenotypes, CAF, CAFS and CAS. Only a few breeds have been studied but CA^F is apparently quite rare. 5. An unidentified protein, designated ‘X’ is present in electrophoretic separations of haemolysates from some sheep. Its presence is dominant to its absence. Polymorphism at this locus is present in all breeds so far studied. 6. A deficiency of reduced glutathione (GSH) in red cells is found in some sheep and is inherited as an autosomal recessive disorder. Sheep with this deficiency have lower red-cell K+ and Na^f concentrations than normal and it is suggested that the HK GSH-deficient sheep may be Ked type sheep. This deficiency has so far only been found with certainty in one breed of sheep. 7. In sheep twin chimeras, admixture of red-cell antigens, haemoglobin and ‘X’ protein types has been found. Various aspects of chimerism, which occurs only rarely in sheep, are discussed. 8. The significance of the genetic variation is discussed in the light of the physiology and immunology of the red cell and of the sheep itself.     

Effects of A23187 and Ca2+ on volume- and thiol-stimulated, ouabain-resistant K+C1- fluxes in low K+ fluxes in low K+ sheep erythrocytes

Ouabain-resistant (OR), C1- -dependent K+ (K+C1-) transport measured by Rb+ influx in isosmotic and anisosmotic media was stimulated by the Ca2+ ionophore A23187 and EGTA (ethylene-glycol-tetracetic acid) in low K+ (LK) but not in high K+ (HK) sheep red cells. Increasing external Ca2+ concentrations, [Ca2+]o, from about 10(-7) to 10(-3)M in presence of A23187 and in absence of EGTA inhibited OR Rb+ influx, in LK red cells osmotically shrunken or swollen as well as treated with the thiol reagent N-ethylmaleimide (NEM). Hence the volume- and the NEM-stimulated K+C1- transport system in LK cells can be experimentally modulated by cellular Ca2+ or other Me2+, which may interact with sites on the K+C1- transporter under the control of membrane sulfhydryl (SH) groups.     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

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.     

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.     

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.     

Passive potassium transport in LK sheep red cells. Effects of anti-L antibody and intracellular potassium

The passive K influx in low K(LK) red blood cells of sheep saturates with increasing external K concentration, indicating that this mode of transport is mediated by membrane-associated sites. The passive K influx, iMLK, is inhibited by external Na. Isoimmune anti-L serum, known to stimulate active K transport in LK sheep red cells, inhibits iMLK about twofold. iMLK is affected by changes in intracellular K concentration, [K]i, in a complex fashion: increasing [K]i from near zero stimulates iMLK, while further increases in [K]i, above 3 mmol/liter cells, inhibit iMLK. The passive K influx is not mediated by K-K exchange diffusion. The effects of anti-L antibody and [K]i on passive cation transport are specific for K: neither factor affects passive Na transport. The common characteristics of passive and active K influx suggest that iMLK is mediated by inactive Na-K pump sites, and that the inability to translocate Na characterizes the inactive pumps. Anti-L antibody stimulates the K pump in reticulocytes of LK sheep. However, anti-L has no effect on iMLK in these cells, apparently because reticulocytes do not have the inactive pump sites which, in mature LK cells, are a consequence of the process of maturation of circulating LK cells. The results also indicate that anti-L alters the maximum velocity of both active and passive K fluxes by converting pumps sites from a form mediating passive K influx to an actively transporting form.     

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

Summary Binding of highly purified¹²⁵I 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 3–6×10³ in HK (MM) and 1.5–4×10³ in LM (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2–1.8×10³ 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 by³H-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.