Incorporation of Na,K-ATPase into human erythrocyte membranes using liposomes

A procedure for incorporation of isolated cattle brain Na,K-ATPase into erythrocyte membranes by proteoliposomes has been elaborated. The Na,K-ATPase activity of proteoliposome-treated human erythrocytes containing incorporated Na,K-ATPase does not exceed that of control erythrocytes. In the erythrocyte membrane the incorporated enzyme exists in a functionally active state and retains the vector properties of the Na+-pump. Exogenous ATP stimulates 22Na influx and 86Rb efflux in and from the erythrocytes.     

Regulatory phosphorylation of FXYD2 by PKC and cross interactions between FXYD2, plasmalemmal Ca-ATPase and Na,K-ATPase

FXYD2 is a regulatory peptide associated with the α-subunit of the kidney Na,K-ATPase. FXYD2 can be phosphorylated by PKA, and its phosphorylation activates Na,K-ATPase. Here we show that FXYD2 is phosphorylated by PKC (PKC-FXYD2-P), by PKA (PKA-FXYD2-P) or by PKA and PKC simultaneously (FXYD2-P₂) modulating both the erythrocyte Na,K-ATPase and the plasma membrane Ca²⁺-ATPase (PMCA). In erythrocyte ghosts, the addition of PKA-FXYD2-P activated Na,K-ATPase by 80%, while non-phosphorylated FXYD2 (np) activated only 55%. The addition of np FXYD2 did not affect PMCA basal activity, but FXYD2-P₂ increased the basal PMCA activity by up to 200%. Calmodulin-activated PMCA activity was increased by np FXYD2 (3-fold) or FXYD2-P₂ (2.5-fold). However, PKC-FXYD2-P increased PMCA activity only by 50%. In contrast, when PMCA was treated with PKA-FXYD2-P, the ATPase activity was inhibited by 50%. The effect of all forms of FXYD2-P on calcium uptake from PMCA resembled the pattern observed in ATP hydrolysis. Our results suggest that the FXYD2 anchoring site could be conserved among the P-ATPase family permitting cross regulation. The effects of FXYD2 on calcium uptake and calcium-stimulated ATP hydrolysis suggest a novel role for FXYD2 on PMCA.     

Characteristics of the Na+-K+-cotransport system in the erythrocyte membrane of patients with hypertension and symptomatic (renal) hypertension

The data on erythrocyte membrane permeability for Na and K ions, obtained in the studies of Na+-K+ cotransport in erythrocytes of 38 patients with essential hypertension, stage I and II, 9 patients with borderline hypertension and 12 patients with symptomatic (renal) hypertension are reviewed. The data demonstrate that Na+-K+ cotransport in Na+ loaded and K+-depleted erythrocytes under the effect of P-chlormercuribenzoate was considerably reduced in patients with essential hypertension and borderline hypertension than in the control group. No deviations from the normal Na+-K+ cotransport were observed in renal hypertension. Disturbances of erythrocyte membrane permeability have been also revealed in practically healthy subjects (15 cases) with family history of hypertension.     

离子对反相HPLC测定人红细胞PRPP合成酶活性

红细胞PRPP合成酶是红细胞内核苷酸代谢的关键酶,它参与嘌呤核苷酸的从头合成与补救合成途径,催化ATP与5-磷酸核糖（R5P）反应生成PRPP与AMP,而PRPP则是嘌呤嘧啶核苷酸合成途径的一个关键性中间产物．Stocchi等[1]采用离子对反相高效液相色谱法（IPrHPLC）测定红细胞内ATP,ADP．AMP含量,曾获得满意的谱峰分离效果．Sakuma等[2]应用rHPLC法测定了正常人及痛风等患者红细胞的PRPP合成酶活性,我们将Sakuma等的测酶技术与IPrHPLC法相结合,改用单液等度洗脱,达到了操作简便和灵敏、准确的要求．1材料和方法1．1试剂…     

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (11-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P_i/mol of -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P_i into the -subunit and the loss of activity of the Na,K-ATPase.     

Glutathionylation of the alpha-subunit of Na,K-ATPase from rat heart by oxidized glutathione inhibits the enzyme

A partially purified Na,K-ATPase preparation from rat heart containing α1- and α2-isoforms of the enzyme was shown to include both subunits in S-glutathionylated state. Glutathionylation of the α1-subunit (but not of the α2-subunit) was partially removed when the preparation was isolated in the presence of dithiothreitol. The addition of oxidized glutathione irreversibly inhibited both isoforms. Inhibition of the enzyme containing the α1-subunit was biphasic, and the rate constants of the inhibition were 3745 ± 360 and 246 ± 18 M^?1·min^?1. ATP, ADP, and AMP protected the Na,K-ATPase against inactivation by oxidized glutathione.     

Comparative characteristics of the properties of erythrocyte Na,K-ATPase in man and the carp Cyprinus carpio

Studies have been made on some of the properties of Na,K-ATPase of a nuclear erythrocytes of man and nuclear erythrocytes of the carp Cyprinus carpio. Human erythrocytes yielded the enzymic activity only after their treatment by a detergent Twin-20; under optimal conditions, it amounted to 2.6 mcmole /ml of erythrocytes per 1 h. In carp erythrocytes, Na,K-ATPase activity could be detected without detergent treatment, being 10-fold higher under optimal conditions than that in human erythrocytes. Repetitive washing of carp erythrocytes from the plasma (for more than 3 times), significantly increased their viscosity and resulted in spontaneous hemolysis. Simultaneously , the activity of Na,K-ATPase increased 2-10 times depending on the composition of incubation media. Under these conditions, the pattern of changes in the enzymatic activity, resulting from shifts in Mg2+ and EDTA concentrations, was altered. The presence of latent Na,K-ATPase activity in the erythrocytes in explained by a low permeability of membranes to ATP and ions. Exogeneous ATP cannot be utilized by the enzyme in the intact human erythrocytes, whereas intact carp erythrocytes exhibit significant permeability to the exogeneous substrate. It is suggested that in vivo this fact may be of physiological importance.     

Na,K-ATPase in dog red cells. Immunological identification and maturation-associated degradation by the proteolytic system

The Na,K-ATPase of red cells from high K+ and low K+ dogs was studied immunologically by using antibodies raised against dog kidney enzyme. Anti-alpha subunit IgGs, which also recognized alpha (+) from brain enzyme, identified the larger subunit of erythrocyte Na,K-ATPase as a homogeneous polypeptide with Mr = 96,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting. In addition, erythrocyte Na,K-ATPase, purified by immunoaffinity chromatography on a monoclonal antibody-coupled column, showed the identity of its polypeptide composition to that of the renal enzyme. Furthermore, it was shown that reticulocyte lysates from high K+ and low K+ dogs substantially degraded 125I-Bolton-Hunter reagent-labeled Na,K-ATPase. This degradation of the enzyme protein was significantly enhanced by the addition of ATP and Mg2+. These results indicate that dog reticulocytes possess some mechanism for protein breakdown involving an ATP-dependent proteolytic system, resulting in the dramatic breakdown of Na,K-ATPase activity during dog reticulocyte maturation into erythrocytes (Maede, Y., and Inaba, M. (1985) J. Biol. Chem. 260, 3337-3343).     

Energetic metabolism during acute stretch-related atrial fibrillation

Background and methods Perturbations in energetic metabolism and impaired atrial contractility may play an important role in the pathogenesis of atrial fibrillation (AF). Besides, atrial stretch is commonly associated with AF. However, the atrial energetics of stretch-related AF are poorly understood. Here, we measured indicators of energy metabolism during acute stretch-related AF. PCr, adenine nucleotides, and derivatives concentrations as well as the activity of the F₀F₁-ATPase and Na,K-ATPase were obtained after 1 h of stretch and/or AF in isolated rabbit hearts and compared to control hearts without stretch and AF. Results After 1 h of stretch-related AF, the total adenine nucleotides’ pool was significantly lower (42.2 ± 2.6 vs. 63.7 ± 8.3 μmol/g protein in control group, P < 0.05) and the PCr/ATP ratio significantly higher (2.3 ± 0.3 vs. 1.1 ± 0.1 in control group P < 0.05), because of ATP, ADP, and AMP decrease and PCr increase. The sum of high-energy phosphate compounds did not change. There were no significant differences in F₀F₁-ATPase nor Na,K-ATPase activity between the groups. Conclusions Results show that in this experimental model, acute stretch-related AF induces specific modifications of atrial myocytes energetics that may play a pivotal role in the perpetuation of the arrhythmia.     

Binding of Na+ ions to the Na,K-ATPase increases the reactivity of an essential residue in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI), a new imidazole-based probe, results in irreversible loss of enzymatic activity. Inactivation of 95% of the Na,K-ATPase activity is achieved by the covalent binding of 1 molecule of [3H]NIPI to a single site on the alpha-subunit of the Na,K-ATPase. The reactivity of this site toward NIPI is about 10-fold greater when the enzyme is in the E1Na or sodium-bound form than when it is in the E2K or potassium-bound form. K+ ions prevent the enhanced reactivity associated with Na+ binding. Labeling and inactivation of the enzyme is prevented by the simultaneous presence of ATP or ADP (but not by AMP). The apparent affinity with which ATP prevents the inactivation by NIPI at pH 8.5 is increased from 30 to 3 microM by the presence of Na+ ions. This suggests that the affinity with which native enzyme binds ATP (or ADP) at this pH is enhanced by Na+ binding to the enzyme. Modification of the single sodium-responsive residue on the alpha-subunit of the Na,K-ATPase results in loss of high affinity ATP binding, without affecting phosphorylation from Pi. Modification with NIPI probably alters the adenosine binding region without affecting the region close to the phosphorylated carboxyl residue aspartate 369. Tightly bound (or occluded) Rb+ ions are not displaced by ATP (4 mM) in the inactivated enzyme. Thus modification of a single residue simultaneously blocks ATP acting with either high or low affinity on the Na,K-ATPase. These observations suggest that there is a single residue on the alpha-subunit (probably a lysine) which drastically alters its reactivity as Na+ binds to the enzyme. This lysine residue is essential for catalytic activity and is prevented from reacting with NIPI when ATP binds to the enzyme. Thus, the essential lysine residue involved may be part of the ATP binding domain of the Na,K-ATPase.     

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP³⁻, eosin²⁻, p-nitrophenylphosphate, and V_max for Na,K-ATPase and K⁺-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na₂SO₄, and NaNO₃ and in 0.1 M Na-acetate, NaSCN, and NaClO₄. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na₂SO₄ ≈ Na-acetate < NaNO₃ < NaSCN < NaClO₄. We treated the influence of NaCl and Na₂SO₄ on K _diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO₃ ⁻ effect was compatible with competitive binding of NO₃ ⁻ and ADP in addition to the general I-effect. K _diss for E·NO₃ was ∼32 mM. Analysis of V_max/K _m for Na,K-ATPase and K⁺-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA     

P2X(7) receptor activation causes phosphatidylserine exposure in human erythrocytes

Activation of cation channels causes erythrocyte phosphatidylserine (PS) exposure and cell shrinkage. Human erythrocytes express the P2X₇ receptor, an ATP-gated cation channel. The two most potent P2X₇ agonists, BzATP and ATP, stimulated PS exposure in human erythrocytes. Other nucleotides also induced erythrocyte PS exposure with an order of agonist potency of BzATP > ATP > 2MeSATP > ATPγS; however neither ADP nor UTP had an effect. ATP induced PS exposure in erythrocytes in a dose-dependent fashion with an EC₅₀ of ∼75 μM. BzATP- and ATP-induced erythrocyte PS exposure was impaired by oxidised ATP, as well as in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X₇ receptor. ATP-induced PS exposure in erythrocytes was not significantly altered in the presence of EGTA excluding a role for extracellular Ca²⁺. These results show that P2X₇ activation by extracellular ATP can induce PS exposure in erythrocytes.     

Angiotensin-converting enzyme and metals in untreated essential hypertension

Hpertension is an important health problem throughout the world and a risk factor for many diseases. Angiotensin-converting enzyme (ACE), a component of the renin-angiotensin system, has an important role in the regulation of blood pressure. Zinc (Zn), a trace element with important biological functions, is located in the catalytic site of ACE. Calcium (Ca), magnesium (Mg), sodium (Na), and potassium (K) also appear to be involved in hypertension pathogenesis. In this study, plasma ACE activities and Ca_t, Ca_i, Mg, Na, K, and plasma/erythrocyte Zn levels of 20 untreated patients with essential hypertension and 28 helthy individuals were evaluated. Plasma ACE activities (p<0.05) and erythrocyte Zn concentrations (p<0.001) were significantly higher in patients with essential hypertension than values of the control group. No significant difference was found between plasma Zn concentrations of the groups (p>0.05). Plasma Ca_t (p<0.001) and Mg levels (p<0.05) in essential hypertension were significantly lower than those of controls. Plasma Na, K, and Ca_i levels remained normal in essential hypertension. There are complex associations between metals and arterial pressure. Ca and Mg deficiencies seem to be associated with increased prevalence of hypertension. Increases in erythrocyte Zn may have a future potential use for diagnosis of hypertension.     

Peripheral resistance and red cell LiNa countertransport in borderline hypertensives

We have studied ouabain-resistant, external sodium-stimulated, lithium efflux (LiNa countertransport) in red blood cells from 21 borderline hypertensives with at least one hypertensive first degree relative (BH-F), 19 borderline hypertensives without family history of essential hypertension (BH-NF), and 35 age-matched normotensive subjects. The data indicate the finding of an increased LiNa countertransport in all BH (F+NF), but with a significant overlap between BH values and control ones: LiNa countertransport is significantly higher only in BH-F but it is normal in BH-NF. Moreover, there is a significant correlation of LiNa countertransport to total peripheral resistance but not to mean blood pressure in all hypertensive patients. It is suggested that in BH the increase of erythrocyte Na flux is mediated by the NaNa exchange diffusion, and its abnormality may be associated to the hereditary trait of essential hypertension rather than the high blood pressure per se, probably resulting in the development of hypertension, through the increased vascular smooth muscle tone.     

Evaluation of intra-erythrocyte Na+ activity in persons at high risk for essential arterial hypertension and as an aid in differential diagnosis from secondary forms of hypertension

The intracellular "Na+ activity" was measured in erythrocytes of normotensive subjects (46), in essential hypertensive patients (18), in their children (20) and in patients with secondary hypertension (8). In normotensive subjects without a genetic trait of hypertension intracellular "Na+ activity" was 7.3 +/- 0.8 mmol/l, in secondary hypertensive patients was 7.5 +/- 0.6 mmol/l, in essential hypertensive patients was 10.9 +/- 1.1 mmol/l and in their children was 8.6 +/- 2.1 mmol/l. In this group (children) it was possible to differentiate between 2 population, the 1 degree with height intracellular "Na+ activity" (8); the 2 degrees with normal intracellular "Na+ activity".     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

Relationship between (Na + K)-ATPase activity, lipid peroxidation and fatty acid profile in erythrocytes of hypertensive and normotensive subjects

Oxidative stress may play a role in the pathogenic mechanism of essential hypertension. Lipid peroxidation can alter the cellular structure of membrane-bound enzymes by changing the membrane phospholipids fatty acids composition. We investigated the relationship between (Na + K)-ATPase activity, lipid peroxidation, and erythrocyte fatty acid composition in essential hypertension. The study included 40 essential hypertensive and 49 healthy normotensive men (ages 35–60 years). Exclusion criteria were obesity, dyslipidemia, diabetes mellitus, smoking, and any current medication. Patients underwent 24-h ambulatory blood pressure monitoring and blood sampling. Lipid peroxidation was measured in the plasma and erythrocytes as 8-isoprostane or malondialdehyde (MDA), respectively. Antioxidant capacity was measured as ferric reducing ability of plasma (FRAP) in the plasma and as reduced/oxidized glutathione (GSH/GSSG ratio) in erythrocytes. (Na + K)-ATPase activity and fatty acids were determined in erythrocyte membranes. Hypertensives had higher levels of plasma 8-isoprostane, erythrocyte MDA, and relative percentage of saturated membrane fatty acids, but lower plasma FRAP levels, erythrocyte GSH/GSSG ratio, (Na + K)-ATPase activity and relative percentage of unsaturated membrane fatty acids, compared with normotensives. Day-time systolic and diastolic blood pressures correlated positively with lipid peroxidation parameters, but negatively with (Na + K)-ATPase activity. These findings suggest that the modulation of (Na + K)-ATPase activity may be associated with changes in the fatty acid composition induced by oxidative stress and provide evidence of a role for this enzyme in the pathophysiology of essential hypertension.     

Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.     

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ + K+, and 5'-adenylylimidodiphosphate

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate. (AMP-P(NH)P. This compound, in which the oxygen connecting the β and γ phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg²⁺-ATPase activities. The $\text{K}{}_{\mathrm{<mtext>1</mtext>}}$ of AMP-P(NH)P for Mg²⁺-ATPase activity was 2.0 · 10^?4 M and, while the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of ATP for this activity was also 2.0 · 10^?4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na⁺, K⁺ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 · 10^?3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-fre porcine erythrocyte ghosts were studied in order to characterize the system more adequately.