Some Kinetic and Metabolic Characteristics of Calcium-Induced Potassium Transport in Human Red Cells

When fresh human erythrocytes or their ghosts are incubated with Ca + IAA (iodoacetic acid) + adenosine, K permeability increases; K permeability also increases when energy-depleted cells or their ghosts are incubated with Ca alone. Na transport decreases or remains unaltered in both situations. The Ca-induced increase in K permeability in the depleted cell system is qualitatively similar to that seen in the fresh cell system and furnishes a means for studying the metabolic dependence of calcium's action. Studies with the depleted system suggest that the normal refractiveness of the cell to calcium is provided by a metabolically dependent substrate. Removal of this substrate allows Ca to enter the cell and exert its effect. By using ⁴⁷Ca, a maximum value was obtained (3–7 x 10^-6 moles/liter of red blood cells) for the quantity of calcium that is taken up by the cell and responsible for the change in K permeability. Measurements of the unidirectional fluxes of K, obtained during the time Ca increases K permeability, appear to satisfy the flux ratio equation for passive diffusion through a membrane.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

Sarcoplasmic reticulum. XVI. The permeability of phosphatidyl choline vesicles for calcium

The Ca permeability of phosphatidyl choline vesicles of diverse fatty acid composition was measured. The rate of ⁴⁵Ca release from liposomes equilibrated with 1 mm⁴⁵CaCl₂ was found to be about 8 × 10⁻¹⁸ moles of Ca/cm²/sec for egg lecithin and about 5.3 × 10⁻¹⁷ moles of Ca/cm²/sec for dioleyllecithin at 30 °. Incorporation of cholesterol into dioleyllecithin micelles reduced the rate of Ca release. The Ca permeability of the phosphatidyl choline micelles was insensitive to changes in the pH, calcium or sodium concentration of the medium but increased with increasing temperature. The effect of temperature was most marked with dioleyl lecithin dispersions, but was clearly apparent with dipalmitoyl, plant, bovine, and egg lecithins as well. The activation energy of Ca release fell in the range of 4.2–9.6 kcal/mole. Macrocyclic antibiotics (valinomycin, tyrocidin, and gramicidin) at relatively high concentration increased the rate of Ca release similarly to their effects on fragmented sarcoplasmic reticulum membranes.     

The release of membrane-bound calcium by radiation and sulfhydryl reagents

Effects of ionizing radiation and of sulfhydryl reagents on the ⁴⁵Ca binding of red cell membranes were studied. Corresponding effects of these agents on potassium leak from intact red cells were also determined. Essentially all the ⁴⁵Ca associated with the ghosts appeared to be bound. Calcium binding could be described by assuming two independent groups of binding sites with dissociation constants of about 6 × 10^?4 m and 2 × 10^?4 m. The total binding capacity was about 2.5 × 10^?4 moles/g ghost protein. Membrane calcium was decreased by radiation and by the two sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and N-ethyl maleimide (NEM). The tightly bound calcium fraction appeared to be most affected by these agents. Changes in potassium leak evoked by varying doses of agents appeared to parallel effects on membrane calcium. These investigations suggest that the increased cation permeability observed after exposure or red cells to radiation or sulfhydryl reagents may be related to alterations in the calcium-binding properties of the cell membrane.     

Passive Ca transport in human red blood cell ghosts measured with entrapped arsenazo III

The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid during the first 5 min after Ca is added to the outside and declining thereafter. The rate of Ca influx is a nonlinear function of extracellular Ca and plateaus as the latter is increased above 1 mM. The rate of Ca influx was measured as a function of the transmembrane gradients of Na and K and changes in the permeability of the membrane to K and Cl produced by valinomycin and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid), respectively. Changes in the rate of Ca influx are consistent with expected effects of these treatments on the membrane potential. Oligomycin (10 micrograms/ml) and quinidine (1 mM) inhibit the rate of Ca uptake by inhibiting Ca-induced changes in the K permeability. At constant membrane potential, furosemide produced a slight (15%) consistent increase in Ca uptake. Other experiments show that resealed ghosts are heterogeneous in their passive permeability to Ca and that A23187 can be used to effectively eliminate such differences. The results of this paper show that resealed human red cell ghosts containing arsenazo III can be used to continuously monitor intracellular free Ca and to study the factors that influence the permeability of the red cell membrane to Ca.     

Nucleotide binding and hydrolysis properties of Neurospora crassa cytosolic molecular chaperones, Hsp70 and Hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.

Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region.     

Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis

A detailed study has been made of the permeability characteristics of human erythrocyte ghosts prepared under isoionic conditions by a glycol-induced lysis (Billah, M.M., Finean, J.B., Coleman, R. and Michell, R.H. (1976) Biochim. Biophys. Acta 433, 45–54). Impermeability to large molecules such as dextran (average molecular weight 70 000) was restored immediately and spontaneously after each of the 5–7 lyses that were required to remove all of the haemoglobin. Permeabilities to smaller molecules such as MgATP^2?, [³H]inositol and [¹⁴C]choline were initially high but could be greatly reduced by incubation at 37°C for an hour. The extent of such resealing decreased as the number of lyses to which the ghosts had been subjected increased. Both removal of haemoglobin and permeabilities to small molecules were affected significantly by pH, Ca²⁺ concentrations and divalent cation chelators. Maximum resealing was achieved in ghosts prepared in the basic ionic medium (130 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 10 mM $\text{N-2-}\text{hydroxyethylpiperazine}\text{-N′-2-}\text{ethanesulphonic}$ acid (HEPES)) at pH 7.0 (0°C) and with a calcium level around 10^?5 M. Acidic pH facilitated the removal of haemoglobin whilst the presence of divalent cation chelators slowed down its release. Retention of K⁺ by ghosts loaded with K⁺ during the first lysis and subsequently incubated at 37° C was substantial but little K⁺ could be retained within the haemoglobin-free ghosts. Permeability of the ghosts to K⁺ after one lysis was affected by temperature, pH, Ca²⁺ concentrations and by the presence of divalent cation chelators.     

Role of membrane-bound Ca in ghost permeability to Na and K

Summary The permeability of red cell ghosts to K is determined by the amount of membrane-bound Mg which, in turn, depends on internal Mg. Contrasting with such effect, an increase in cellular Ca raises K permeability. To test whether this, action is due to a competitive displacement of membrane Mg, the free Ca content of human red cell ghosts was altered by means of Ca-EGTA buffers. Net Na and K movements as well as Ca and Mg bindings, were assessed after incubation in a Na-medium at 37°C. Raising Ca from 3×10^–7 to 1×10^–2M caused a large K efflux with very little Na gain. Under similar conditions, Ca binding was increased without affecting membranebound Mg. Both Ca binding and K loss were markedly diminished by either adding ATP to the hemolytic medium or increasing internal Mg at a fixed Ca concentration. A Scatchard analysis showed three Ca binding sites, two of them having high affinity. It is concluded that Ca action does not arise from a displacement of membrane-bound Mg but from binding to different sites in the membrane. Presumably, high affinity sites are involved in the control of K permeability.     

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of 5-carbomethoxyvaleramidine during hydrolysis of the protein cross-linking agent dimethyl adipimidate

Imidoesters have been used in biological studies to measure interresidue distances of proteins and macromolecular complexes, and in hematology as antisickling agents. Treatment of human red blood cells with¹⁴C-labeled dimethyl adipimidate (DMA), a bifunctional imidoester with antisickling properties, was followed by gradual loss of radioactivity from the treated cells. The radioactive compound released was isolated by thin-layer chromatography and identified by high-resolution mass spectrometry and by carbon-13 nuclear magnetic resonance, ultraviolet, and infrared spectroscopy as 5-carbomethyoxyvaleramidine, which was also shown to be the major product of DMA hydrolysis in vitro at physiologic pH in phosphate buffer. High-resolution mass spectrometry studies indicated that this product is formed via cyclization to a reactive intermediate (7-methoxy-2-imino-3,4,5,6-tetrahydro-2H-azepine) followed by hydrolysis. The intermediate exhibited strong UV absorbance, maximal at 232 nm. Such an intermediate would be capable of participating in cross-linking reactions which would have smaller dimensions than those observed with the imidoester in its extended form. The hydrolysis product, an unreactive species, should have no toxic effects on individuals receiving infusions of DMA-treated red cells.     

Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes

The relationship between active extrusion of Ca⁺⁺ from red cell ghosts and active uptake of Ca⁺⁺ by isolated red cell membrane fragments was investigated by studying the Ca⁺⁺ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca⁺⁺ adenosine triphosphatase (ATPase) and ATP-dependent Ca⁺⁺ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more ⁴⁵Ca in the presence of ATP compared to preparation B. Separation of the ⁴⁵Ca-labeled membrane vesicles by density gradient centrifugation showed that the ⁴⁵Ca label was localized to the inside-out vesicle fraction. In addition, the ⁴⁵Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of ⁴⁵Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La⁺⁸ (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca⁺⁺ by red cell membrane fragments is due to the active transport of Ca⁺⁺ into inside-out vesicles.     

A method for estimating free Ca within human red blood cells,with an application to the study of their Ca-dependent K permeability

Summary Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600–6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 m Ca in fresh cells, and from 0.4 to 0.7 m Ca in blood-bank cells, at pH 7.2 and 37°C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using⁸⁶Rb as a tracer for K.⁸⁶Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3–6 times faster in ghosts than in cells.     

Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.     

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Measurement of the filtration coefficient (Kfc) in the lung of Gallus domesticus and the effects of increased microvascular permeability

The filtration coefficient (Kfc) is a sensitive measure of microvascular hydraulic conductivity and has been reported for the alveolar lungs of many mammalian species, but not for the parabronchial avian lung. This study reports the Kfc in the isolated lungs of normal chickens and in the lungs of chickens given the edemogenic agents oleic acid (OA) or dimethyl amiloride (DMA). The control Kfc =0.04±0.01 ml min⁻¹ kPa⁻¹ g⁻¹. This parameter increased significantly following the administration of both OA (0.12±0.02 ml min⁻¹ kPa⁻¹ g⁻¹) and DMA (0.07±0.01 ml min kPa⁻¹ g⁻¹). As endothelial cadherins are thought to play a role in the dynamic response to acute lung injury, we utilized Western blot analysis to assess lung cadherin content and Northern blot analysis to assess pulmonary vascular endothelial (VE) cadherin expression following drug administration. Lung cadherin content decreases markedly following DMA, but not OA administration. VE cadherin expression increases as a result of DMA treatment, but is unchanged following OA. Our results suggest that the permeability characteristics of the avian lung are more closely consistent with those of the mammalian rather than the reptilian lung, and, that cadherins may play a significant role in the response to acute increases in avian pulmonary microvascular permeability.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

The sodium and potassium activated ATPase. II. Comparative study of intestinal epithelium and red cells

The Na-K ATPase found in sedimentable fractions of intestinal epithelium of rats hydrolyzed cytidine triphosphate nearly as well as ATP (25% to 50%); was active only in presence of divalent cations, with specificity for Mg (100%), Mn (50%) and Ca (10%); showed a plateau of activation when Mg concentrations were in excess of substrate; and was inhibited by a second divalent cation (Zn > Mn > Ca), and by 3 × 10^?4 M ouabain (50%). Parallel assays of rat red cell ghosts showed differences in substrate specificity (CTP was not utilized), in activation kinetics (activation peak with Mg) and in greater specificity to Mg (Mn was a weaker activator and Zn was a weaker inhibitor). Stabilities also differed in the two preparations: Na? K ATPase of intestinal epithelium was activated by sucrose extraction and denatured during cytolysis at room temperature, while that of red cell fragments was denatured during sucrose extraction and preserved by hemolysis at room temperature. Other properties of Na? K ATPase studied in the two tissues included activation by monovalent cations (optimum at 160 mM Na, 15 mM K), specificity to monovalent cations, and sensitivity to lipid solvents and to some drugs. The data were discussed in terms of comparative properties of Na? K ATPases of various cells. Residual ATPase activities of intestinal epithelium and red cell ghosts were shown to differ in substrate specificity, inhibition and activation. “Residual ATPase” from intestinal epithelium was a zinc-activated nucleoside polyphosphate phosphohydrolase, while ghosts contained Mg? ATPase. Only the latter enzyme was specific to ATP and Mg, activated by Ca in presence of Mg, and sensitive to inhibition by PCMB and Zn.     

Changes in Amino Acid Transport During Red Cell Maturation

We studied amino acid transport in sheep red blood cells (RBCs) as a function of cell maturation. Transport of amino acids is decreased strikingly in the mature mammalian RBC compared to the immature reticulocyte. Blood obtained 5-6 days after massive bleeding was fractionated on dextran gradients. In the mature erythrocyte amino acids are taken up only slowly, and in the normal experimental interval (60 min) the concentration in the cell does not reach that of the medium. In contrast, the reticulocyte-rich (top) fraction (50-90% reticulocytes) accumulates certain amino acids, particularly histidine, methionine, and leucine. The underlying process is ATP-independent and Na⁺-insensitive, and has properties consistent with exchange diffusion, i.e., accelerated uptake or efflux when unlabeled solute is present on the trans side. The process is apparent not only in intact cells but also in resealed ghosts. The decrease in activity of amino acid transport is a function of red cell maturation. Thus it can be shown that (a) separation of cells according to their density 1, 2, and 3 weeks after bleeding leads to progressively lower amino acid transport activity with increasing cell density; and (b) during in vitro long-term incubation at 37°C of reticulocyte-rich, unfractionated blood (5–10% reticulocytes), amino acid transport decreases while red cell integrity is maintained, as evidenced by the retention of a normal K⁺ gradient and the absence of hemolysis. The progressive loss is seen with resealed ghosts as well as with intact cells. Not all the amino acids examined participate in this exchange process. The most actively exchanged are histidine, leucine, methionine, and phenylalanine. Glycine, proline, arginine, and a-amino isobutyric acid do not participate in the exchange process.     

Inhibition of K+-sensitive p-nitrophenylphosphatase activity on rhesus-positive red cells after incubation with IgG-anti-rhesus-D

The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K⁺-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na⁺ + K⁺)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K⁺-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl₃ at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells.