A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells.

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K+ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K+-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K+ genotype of the sheep and was not associated with either "high K+"- or "low K+"-type Na+ pump. No Ca2+-dependent change in K+ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K+ channel in the mammalian red cell and the relationship between the K+ channel and the Na+ pump are 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.     

Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation

Influx of the K⁺ analogue Rb⁺ was measured through the ouabain-sensitive Na⁺/K⁺ pump and the ouabain-insensitive “leak” pathways in Cl^? or NO in mature red cells from adult pigs and in reticulocytes naturally occurring in 7-day-old piglets. In reticulocytes, Rb⁺ influxes by the two pathways were of about equal magnitude in Cl^? (13 and 10 mmoles/liter cells × hr) and at least 25-fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na ⁺ media, a portion of the ouabain-insensitive “leak” flux of Rb⁺ was Cl^? dependent (Rb⁺Cl^? transport) as NO replacement reduced Rb⁺ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N-ethylmaleimide (NEM) stimulated Rb⁺Cl^? transport about twofold in reticulocytes and up to 13-fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain-sensitive Rb⁺ pump and ouabain-insensitive Rb⁺Cl^? influx were lost. In contrast, the NEM-stimulated Rb⁺Cl^? transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb⁺Cl^? transport. Although the experimental variability precluded a full assessment of significant changes in the small Na⁺/K⁺(Rb⁺) pump and Rb⁺Cl^? fluxes in mature pig red cells kept for the same time period in vitro, Rb⁺ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na⁺/K⁺ pump and Cl^?-dependent K⁺ fluxes in vivo.     

Different red cell populations in newborn, genetically low potassium sheep: relation to hematopoietic, immunologic and physiologic differentiation

Three red cell populations have been distinguished in genotypically low potassium (LK) newborn sheep by an improved electrical sizing method and were best approximated by a logarithmic normal distribution. Labeling studies with ⁵¹Cr and ⁵⁹Fe exclude transformation of the three red cell populations into each other. Population I, consisting of large red cells (mean volume 36 μm³), with a comparatively slow electrophoretic mobility is present at birth and disappears within three to four weeks from circulation. These cells possess a high potassium (HK) steady state concentration, a K⁺ pump influx activity at least 5-fold greater than observed in adult LK red cells, very low amounts of the L antigens generally associated with the LK property, and do not respond to the stimulatory action of the L antibody. The first population is gradually replaced by population II comprising small red cells (mean volume 28 μm³) of intermediate electrophoretic mobility and with a peak production around day 20 after birth. The potassium concentration, [K⁺]_c, in these cells appears to be lower than in the cells of population I but the L antigen content is increased. Formation of population III (mean volume 30 μm³ and comparatively fast electrophoretic mobility) follows closely that of population II and is preceded by a sharp increase in reticulocytosis. The red cells of population III exhibit parameters characteristic for adult LK cells: low [K⁺]_c and K⁺ pump activity, fully developed L antigen content, and an almost maximal response to the K⁺ pump stimulating effect of anti-L. In L and M antigen positive LK red cells of newborn sheep, the development of the M antigen parallels that of the L antigen. The data are consistent with the hypothesis that cellular replacement and not maturation is the major factor in controlling the HK-LK transition in newborn sheep.     

Active potassium transport in reticulocytes of high-K and low-K sheep

The kinetics of active K⁺ transport were studied in immature red blood cells cells from high-K⁺ and low-K⁺ sheep, particularly with respect to the effects of varying intracellular K⁺ concentration, [K]_i. Comparison was made with active transport, or pump, activity in mature high-K⁺ and low-K⁺ red cells. Reticulocytes from both types of sheep had much higher maximal active K⁺ influxes than did mature cells. In both types of reticulocytes, and in mature high-K⁺ cells as well, the pump was relatively insensitive to increasing [K]_i. In contrast, intracellular K⁺ markedly inhibited the pump in mature low-K⁺ cells. Active K⁺ transport in low-K⁺ reticulocytes, however, as in mature low-K⁺ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K⁺ pumps of high-K⁺ and low-K⁺ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K⁺ and low-K⁺ cells with K⁺ pumps of very different kinetic characteristics.     

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.     

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na⁺ concentrations and water content. Such cells provided an experimental model to study the effect of Na⁺/K⁺ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na⁺/K⁺ pump by elevated intracellular Na⁺ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na_out⁺ : 2 K_in⁺ stoichiometry of the Na⁺/K⁺ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.     

Cation transport in different volume populations of genetically low K+ lamb red cells

During the first three months after birth lambs produce sequentially three erthryocyte populations of different mean volume as demonstrated by electric sizing methods (Valet, Franz, and Lauf, J. Cell. Physiol. 94 (1978) 215). We separated by centrifugal elutriation the small volume population (type II) red cells of a genotypically low K⁺ (LK) lamb from the population containing the larger volume type I and III cells, an admixture of fetal (I) and adult (III) erythrocytes. The cells were separated at various time intervals after birth and analyzed with respect to their volumes, cation contents, and cation flux properties by means of ⁸⁶Rb uptake. The effect of anti-L on K⁺ pump and leak fluxes was ascertained in unseparated and separated red cells. It was found that the small red cells of population II, transiently present for several weeks, were fully developed LK cells with K⁺ pumps responding characteristically to the stimulatory action of anti-L. In constrast, the larger cells of population I and III were of high K⁺ (HK) nature at early time points, the K⁺ pump activities approximately ten times higher than adult LK cells. These cells constitute an admixture of type I fetal HK cells, and type III reticulocytes which are precursors for the final type III adult LK cells, since anti-L had a small stimulatory effect. At later times, however, only adult type III LK cells predominated. The data directly support our earlier finding that the HK-LK transition in genotypically LK lambs is primarily governed by cellular replacement.     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

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.     

An impurity in phenol red opens an ion channel in cultured human cells

Summary Human fibroblast, bladder carcinoma, and breast carcinoma cells in commercial serum-free media or balanced salt solutions rapidly lose K⁺ and gain Na⁺. This rapid K⁺ loss is caused by one or more impurities in phenol red. Adding serum or albumin to media or to balanced salts prevents K⁺ loss. Quinine also prevents part of this loss in fibroblasts and breast carcinoma cells, suggesting that the impurity acts on an ion channel.     

Potassium-activated phosphatase from human red blood cells

Summary The cell membrane K⁺-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K⁺-free ghosts are suspended in K⁺-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K⁺-rich ghosts suspended in K⁺-free media. These results, together with the fact that Na⁺ competitively inhibits the effects of K⁺ only when present externally, show that the K⁺ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg⁺⁺ requirements for K⁺ activation of the membrane phosphatase are fulfilled by internal Mg⁺⁺. Addition of intracellular Na⁺ to ATP-containing ghosts raises the apparent affinity of the enzyme for K⁺, suggesting that the sites where ATP and Na⁺ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na⁺–K⁺-ATPase system.The authors are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Ion and water shifts produced by ammonium chloride in high K and low K red cells

The effect of ammonium chloride on the cellular Na⁺, K⁺ and water has been examined in human and horse (high K), cow (medium K) and cat (low K) red cells. It was found that high K red cells, especially those of the horse, gained water an Na⁺, whereas the net movement of K⁺ was negligible. There was a correlation between the increase of cellular Na⁺ concentration and of the packed red cell volume. In contrast, the packed cell volume of low K red cells increased slightly or not at all, and Na⁺ ions leaked out from the cells. The high K cells had a lower Cl^? concentration and higher buffer capacity than the low K cells. The results obtained with the medium K (cow) cells usually lay between those of the other two cell types. In all the cases both the plasma and cell pH decreased resulting from the addition of ammonium chloride. The mechanism of movements of water and Na⁺ ions in high K cells remained unsolved, but the response of low K cells to ammonium chloride was near that of a cation exchange resin.     

Ouabain-insensitive sodium/lithium exchange and the effect of anti-L in low potassium sheep erythrocytes

The activity of the ouabain-insensitive Na⁺/Na⁺ exchange system was assessed by measurements of Li⁺ net-uptake in LK and HK sheep erythrocytes in the absence and presence of the L-antibody and various inhibitors. N-ethylmaleimide, p-chloromercuribenzoic sulfonate and phloretin inhibited the exchange by about 50%. Anti-L, while stimulating the K⁺ pump flux in LK cells, did not alter Na⁺/Li⁺ countertransport. The activity of the exchange system with fully saturated internal and external loading sites was estimated to be identical in LK and HK sheep red cells. Hence the Na⁺/Na⁺ exchange system seems to be molecularly unrelated to the ouabain-sensitive Na⁺K⁺ pump in these cells and not under genetic control of the HK/LK and M/L genes.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

On the Concept of Resting Potential—Pumping Ratio of the Na+/K+ Pump and Concentration Ratios of Potassium Ions Outside and Inside the Cell to Sodium Ions Inside and Outside the Cell

In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na⁺/K⁺ pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na⁺/K⁺ pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na⁺/K⁺ pump to prove that the three Na⁺ to two K⁺ pumping ratio of the Na⁺/K⁺ pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na⁺/K⁺ pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is ?30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na⁺/K⁺ pump and the ion concentration ratios play a role in the evolution of animal cells.     

Studies on lithium transport across the red cell membrane

Summary Ouabain-resistant Na⁺–Li⁺ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li⁺ for Na⁺ in a ratio of 11, was present in all four species. Li⁺ uptake by the exchange system increased 30-fold in the order man +–Na⁺ exchange in these species, but bears no relation to the Na⁺–K⁺ pump activity. The activity of the Na⁺–Li⁺ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li⁺ and Na⁺ were similar among the species and individuals (half saturation of the external site at about 1mm Li⁺ and 50mm Na⁺, respectively).50–60% of Na⁺–Li⁺ exchange was blocked by N-ethylmaleimide in all species.p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60–80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li⁺ unaltered. Phloretin was a potent inhibitor in all species. 1mm each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na⁺–Li⁺ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na⁺–Na⁺ exchange in beef erythrocytes.It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na⁺ for Na⁺, Na⁺ for Li⁺, or Li⁺ for Li⁺. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na⁺–K⁺ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na⁺ transport systems described in the literature.     

Effects of haemoglobin O2 saturation on volume regulation in adrenergically stimulated red blood cells from the trout, Oncorhynchus mykiss

Adrenergic stimulation of trout red blood cells activates a Na⁺/H⁺-exchange. If unopposed, the ensuing increase in cell Na⁺ leads to an isosmotic cell swelling. In this study the effect of the level of haemoglobin O₂ saturation on volume regulation has been investigated in adrenergically stimulated red blood cells from trout: at full haemoglobin O₂ saturation, net influx of Na⁺ through the Na⁺/H⁺-exchanger was balanced by net efflux of K⁺ and no increases in cell volume took place. In contrast, at low O₂ saturation (8–14%) adrenergic stimulation led to a substantial increase in cell Na⁺, K⁺ and volume. Moreover, cell volume recovery after adrenergic swelling was incomplete at low O₂ saturation, whereas cells at high O₂ saturation exhibited a fast and complete cell volume recovery. In cells exposed to alternating high and low O₂ saturation, volume regulation was similar to the regulation found in cells maintained at high O₂ saturation. In cells at high O₂ saturation, extrusion of cellular Na⁺ by the Na⁺/K⁺-pump significantly contributed to the volume decrease. It is concluded that trout red blood cells at high or alternating O₂ saturations possess a powerful regulatory volume decrease response that is shut off at low O₂ saturation. The physiological implications of this regulation is discussed. Accepted: 30 September 1996     

Ouabain Binding and Potassium Transport in Young and Old Populations of Human Red Cells

Human red blood cells were separated according to density by centrifugation through mixtures of phthalate esters. The densest 20% of the erythrocyte population (old cells) had reduced volume and water content compared to the lightest 20% of the cells (young cells). Corpuscular hemoglobin content was unchanged. Young cells had 50% more potassium (K⁺) than old cells, but their total intracellular concentration was only slightly higher; old cells had a small increase in sodium (Na⁺) concentration. Active K⁺ transport of young cells was 37% higher than that of old cells. [³H] + Ouabain binding revealed that this difference was the result of more K⁺ pump sites on young cells, which bound 530 ouabain molecules per cell at 100% K⁺ pump inhibition, as compared to 400 for old cells; unseparated cells bound 450-500 molecules. The relative rates of ouabain binding were identical for the two cell types. Old cells exhibited a greater passive permeability to K⁺, haying a rate coefficient for ouabain-insensitive K⁺ influx 1.8 times that of young cells. There is evidence to suggest that in the face of reduced pump activity this augmented K⁺ “leak” might enhance the osmotic stability of the old cells and function to lengthen their life span.     

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.