Recognition of sodium- and potassium-dependent adenosine triphosphatase on mouse lymphoid cells by means of a monoclonal antibody

Summary Previous evidence has established the similarity between (Na⁺+K⁺)-ATPase (ATP phosphohydrolase, EC.3.6.1.3) and the antigen recognized by the rat antimouse monoclonal antibody anti-BSP-3. This antibody has been used for investigation of the surface expression and biochemical analysis of the enzyme in different mouse lymphoid populations. The BSP-3 determinant is found on almost all thymocytes and concanavalin A-induced thymocytes, to a lesser extent on bone marrow cells and also on a minor population of spleen cells. Spleen cells from athymic mice are negative. The (Na⁺+ K⁺)-ATPase purified from mouse thymus by affinity chromatography migrates in SDS-polyacrylamide gels in the form of two polypeptide chains of 105000 and 51000 daltons. Chains of the same molecular weight, fractionated on SDS-PAGE from microsomes of mouse thymuses, are shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Immunoprecipitation with anti-BSP-3 from surface iodinated thymocytes yields only the small subunit. Comparison of the chains isolated from thymus and brain shows molecular weight differences in both subunits. These results, and variations in the reactivity pattern of the anti-BSP-3 antibody on several cell types, may indicate a possible heterogeneity of the (Na⁺+K⁺)ATPase expressed by various tissues and cells.     

Properties of the housefly head sodium- and potassium-dependent adenosine triphosphatase

The properties are described of a solubilised Na⁺- and K⁺-dependent ATPase prepared from frozen housefly heads. The affinity for ATP and the requirements for Na⁺, K⁺, and Mg²⁺ of the flyhead enzyme are similar to those of other animal Na⁺- and K⁺-dependent ATPases. Ouabain appears to inhibit the insect enzyme by interaction at the K⁺-binding site in a non-competitive manner.The solubilised ATPase contained a p-nitrophenylphosphatase activity which showed a requirement for K⁺ and Mg²⁺ and which was inhibited by ouabain.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.     

Organ secificity of rat sodium- and potassium-activated adenosine triphosphatase.

We compared several Na,K-ATPase preparations from various organs of the rat. The brain Na,K-ATPase differed from the enzymes of other organs in its pH dependence and responses to ouabain and N-ethylmaleimide in spite of similarities in the kinetic parameters of activation by Na+, K+, Mg2+, and ATP. The optimum pH of the brain MaI-enzyme was at 7.4 to 7.5 at 37 degrees D. The Lubrol extract of this brain enzyme preparation showed a lower optimum oH of 6.6. When the Lubrol extract of the brain was fractionated wtih (NH4)2SO4, the activity of the precipitate in the neutral pH region was restored. On the other hand, the optimum pH of the kidney NaI-enzyme was slightly affected by Lubrol and ammonium sulfate treatments (pH 7.5 leads to 7.3). The brain enzyme (K 1/2 = 0.9 microM) showed about 100-fold higher sensitivity to ouabain than the enzymes from other organs (I 1/2 = 100 microM) in the presence of 120 mM Na+ and 10 mM K+. In a Hill plot of the ouabain inhibition, the former failed to give a linear relationship, while the latter gave a straight line with a Hill coefficient of 1.0. The effect of K4 on the brain enzyme-ouabain interaction led us to consider that the brain enzyme might have two components as regards ouabain affinity, high and low affinity components. The time course of N-ethylmaleimide inhibition of the brain enzyme was rapid and biphasic, while the kidney enzyme showed only a slow phase following pseudo-first order kinetics. ATP protected the kidney enzyme activity completely agai,st N-ethylmaleimide inhibition, but the protection of the brain enzyme activity by ATP was only partial. We divided rat Na,K-ATPases into two groups, the brain type, which is restricted to the central nervous system, and the kidney type, which is found in most organs.     

Conformational change of sodium- and potassium-dependent adenosine triphosphatase. Conformational evidence for the Post-Albers mechanism in Na+- and K+-dependent hydrolysis of ATP

The addition of ATP with K+ to pig kidney Na+,K+-ATPase (EC 3.6.1.3) modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide induced a transient decrease (t 1/2 = 0.01 s) in the fluorescence in the presence of Mg2+ with 0.64 M Na+, followed by a slow increase (t 1/2 = 0.08 s), to give a higher steady level than that observed without K+. The addition induced a transient increase (t 1/2 less than 0.02 s) in the amount of phosphoenzyme, followed by a slow decrease (t 1/2 = 0.08 s), but the addition without K+ induced a monophasic increase (t 1/2 = 0.02 s). The addition of ATP in the presence of 2 M Na+ with Ca2+ induced a monophasic decrease (t 1/2 = 0.1 s) in the fluorescence along with a much slower increase (t 1/2 = 1.2 s) in the amount of phosphoenzyme. No significant burst of acid-labile phosphate was observed. The data showed clearly the accumulation of the enzyme-ATP complex preceding the phosphoenzyme formation. Fluorescence intensity of these enzyme species and the amount of phosphoenzyme permitted the simulation using the reaction mechanism including enzyme-ATP complex, ADP-sensitive phosphoenzyme, K+-sensitive phosphoenzyme, and K+-bound enzyme. The simulation gave a good fit to the experimental data which showed that ATP is hydrolyzed in sequence through the above intermediates in the presence of both Na+ and K+.     

Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase.

In order to study the action of the divalent cation which is essential for phosphorylation of sodium- and potassium-transport adenosine triphosphatase, magnesium ion, the normal ligand, was replaced with calcium ion, which had properties diffeerent from those of Mg2+, Mn2+, Fe2+, Co2+, Ni2+, or Zn2+. Phosphorylation of the enzyme from ATP at pH 7.4 in the presence of Na+ and Ca2+ yielded a Ca.phosphoenzyme (60% of the maximal level) with a normal rate of dephosphorylation following a chase with unlabeled Ca.ATP (PK = 0.092S-1 at 0 degrees C). In contrast, after a chase by a chelator, namely ethylenediaminetetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic acid, or ethylene glycol bis-(beta-aminoethyl ether)N,N'-tetraacetic acid, dephosphorylation slowed within 5 s and half of the initial phosphoenzyme remained with a stability about 5-fold greater than normal. Three states of the phosphoenzyme were distinguished according to their relative sensitivity to ADP or to K+ added during a chase. Normally prepared Mg.phosphoenzyme was sensitive to K+ but not to ADP; Ca.phosphoenzyme was sensitive either to ADP or to K+; and the stabilized phosphoenzyme prepared from Ca.phosphoenzyme by addition of a chelator was sensitive neither to ADP nor to K+ nor to both together. Addition of Ca2+ to the stabilized phosphoenzyme restored the reactivity to that of Ca.phosphoenzyme. Addition of Mg2+ to the stabilized phosphoenzyme changed the reactivity to that of Mg.phosphoenzyme. Therefore, this unreactive, stabilized state of the phosphoenzyme appeared to be a divalent cation-free phosphoenzyme. With respect to sensitivity to ouabain, Ca.phosphoenzyme was as sensitive as Mg.phosphoenzyme but calcium-free phosphoenzyme was much less sensitive. It was concluded that the divalent cation required for phosphorylation normally remains tightly bound to the phosphoenzyme and is required for normal reactivity. Calcium ion was almost unique in dissociating relatively easily from the phosphoenzyme. Strontium ion appeared to act similarly to Ca2+.     

Specific sodium-22 binding to a purified sodium + potassium adenosine triphosphatase. Inhibition by ouabain.

Analysis of sodium-22 binding to purified sodium + potassium ion-activated adenosine triphosphatase (Na+, K+)-ATPase reveals the presence of two classes of binding sites. The higher affinity site (Kd = 0.2 mM) binds 6 to 7 nmol of sodium per mg of protein. Pretreatment of (Na+, K+)-ATPase with ouabain blocks the binding of sodium to this higher affinity site. Neither heat-denatured enzyme nor phospholipids extracted from the (Na+, K+)-ATPase contain a ouabain-inhibitable, higher affinity sodium binding site. The ouabain enzyme complex therefore appears to contain altered binding sites for cations.     

Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody

A monoclonal antibody designated anti-Cl was obtained from a hybridoma clone isolated from a fusion of NS1 myeloma with spleen cells from BALB/c mice injected with homogenate of white matter from bovine corpus callosum. In the adult mouse neuroectoderm, C1 antigen is detectable by indirect immunohistology in the processes of Bergmann glial cells (also called Golgi epithelial cells) in the cerebellum and of Muller cells in the retina, whereas other astrocytes that express glial fibrillary acidic protein in these brain areas are negative for C1. In addition, C1 antigen is expressed in most, if not all, ependymal cells and in large blood vessels, but not capillaries. In the developing, early postnatal cerebellum, C1 antigen is not confined to Bergmann glial and ependymal cells but is additionally present in astrocytes of presumptive white matter and Purkinje cell layer. In the embryonic neuroectoderm, C1 antigen is already expressed at day 10, the earliest stage tested so far. The antigen is distinguished in radially oriented structures in telencephalon, pons, pituitary anlage, and retina. Ventricular cells are not labeled by C1 antibody at this stage. C1 antigen is not detectable in astrocytes of adult or nearly adult cerebella from the neurological mutant mice staggerer, reeler, and weaver, but is present in ependymal cells and large blood vessels. C1 antigen is expressed not only in the intact animal but also in cultured cerebellar astrocytes and fibroblastlike cells. It is localized intracellularly.     

Recognition of a genus-wide antigen ofLegionella by a monoclonal antibody

A monoclonal antibody was produced against a cytoplasmic membrane protein that appears to be common to all species of the genusLegionella. The antibody was positive in polyacrylamide gel electrophoresis and Western blotting with extracts of all of 22 species type strains ofLegionella that were tested. The apparent molecular mass of the protein varied from 57.2 to 62.1 kilodaltons for the 23 species type strains ofLegionella. An enzyme-linked immunosorbent assay (ELISA) was developed with the monoclonal antibody to enable rapid screening of clinical and environmental isolates forLegionella. All of 23 species type strains ofLegionella that were tested were strongly positive with the monoclonal antibody in the ELISA. Among 27 other bacterial species and 84 strains that were tested, onlyBordetella ssp. andAcinetobacter lwoffii were cross-reactive in the ELISA. These two cross-reactive species are readily distinguishable fromLegionella by culture characteristics. The monoclonal antibody may also be useful in tests to detect the genus-wide antigen in body fluids of patients with legionellosis.     

Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed.     

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.     

Phosphorylation from adenosine triphosphate of sodium- and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme.

The relative effectiveness of the ligands Mg2+, Na+, and ATP in preparing sodium plus potassium ion transport adenosine triphosphatase for phosphorylation was studied by means of a rapid mixing apparatus. Addition of 2 mM MgC12, 120 mM NaC1, and 5 muM [gamma-32P]ATP simultaneously to the free enzyme gave an initial phosphorylation rate of about 0.3 mu mol-mg-1-min-1 at 25 degrees and pH7.4. Addition of Mg2+ to the enzyme beforehand, separately or in combination with Na+ or ATP, had little effect on the initial rate. Addition of Na+ only to the enzyme beforehand increased this rate 1.5- to 3-fold. Early addition of ATP 130 ms before Na+ plus Mg2+ increased the rate 6- to 7-fold. Early addition of Na+ plus ATP was most effective; it increased the rate about 10-fold. The data indicate that Na+ and ATP bind in a random order and that each ligand potentiates the effect of the other. The rate of dissociation of ATP from the enzyme was estimated by a chase of unlabeled ATP of variable duration. This rate was slowest in the presence of Mg2+ (k = 540 min-1), most rapid in the presence of Na+ (k = 2000 min-1), and intermediate (k = 1100 min-1) in the absence of metal ions. The effect of Na+ concentration on the rate of phosphorylation was estimated when Na+ with Mg2+ was added to the enzyme-ATP complex. The rate followed Michaelis-Menten kinetics with a maximum of 2.9 mu mol-mg-1 and a Km of 8 mM. The effect of Na+ concentration was also estimated on the increment in the rate of phosphorylation produced by the presence of Na+ with the enzyme-ATP complex beforehand. The increment followed the same kinetics with a maximum of 3.75 mu mol-mg-1-min-1 and a Km of 5.4 mM. In both cases estimation of the Hill coefficient failed to show cooperativity between binding sites for Na+. In contrast, the dependence of ouabain-sensitive ATPase activity on Na+ concentration in the absence of K+ indicated two sites for Na+ with apparent Km values of 0.16 and 8.1 mM, respectively.