Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.     

Photoaffinity labeling of the sodium- and potassium-activated adenosinetriphosphatase with a cardiac glycoside containing the photoactive group on the C-17 side chain

The synthesis and properties of a radiolabeled glycoside photoaffinity probe, [3H]-(3 beta,5 beta,14 beta, 20E)-24-azido-3-[(2,6-dideoxy-beta-D-ribo-hexopyranosyl) oxy]-14-hydroxy-21-norchol-20(22)-en-23-one, containing the photoactive group at the C-17 side chain of the steroid moiety are reported. The molecule binds to the sodium- and potassium-activated adenosinetriphosphatase from porcine kidney outer medulla under type II binding conditions [5 mM MgCl2, 3 mM phosphate, 2 mM ethylenediaminetetraacetic acid, 30 mM tris(hydroxymethyl)aminomethane, pH 7.2, 37 degrees C] in the dark with an equilibrium dissociation constant of (1.4 +/- 0.3) X 10(-7) M. Ultraviolet irradiation of a solution of enzyme plus 3H-labeled probe, followed by analysis of covalently incorporated radiolabel, shows ouabain-displaceable labeling exclusively of the alpha subunit of the sodium- and potassium-activated adenosinetriphosphatase. These data indicate that the binding site of the C-17 side group of cardiac glycosides is located on or near the alpha subunit of this enzyme.     

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.     

Duality of alpha-subunit of canine kidney sodium- and potassium-activated adenosinetriphophatase in electrophoresis

Sodium- and potassium-activated adenosinetriphosphatase (Na+, K+-ATPase) purified from dog kidney outer medulla was examined by polyacrylamide gel electrophoresis and by photoaffinity labeling with N-(ouabain)-N'-(2-nitro-4-azidophenyl)-ethylenediamine (NAP-ouabain). The large subunit band (alpha-band) split into two bands on the gel after the enzyme was heat-treated in the presence of 1% sodium dodecylsulfate (SDS). Of the two bands (alpha I and alpha II), alpha I had the same electrophoretic mobility as the original band, while alpha II moved slightly faster. Total conversion into alpha II was not observed, about half of the original remaining as alpha I. Below 60 degree C, heat treatment did not produce alpha II. Phenylmethylsulfonyl fluoride did not prevent the appearance of alpha II. Both alpha I and alpha II were labeled with [3H]NAP-ouabain. Nonspecific incorporation of [3H]NAP-ouabain also occurred irrespective of illumination, but it was removed either by diffusion during staining and destaining of the gel or by treatment of the enzyme with trichloroacetic acid. It is tentatively concluded that the splitting of the band reflects some intrinsic differences in situ of the alpha-subunit of dog kidney membrane Na+,K+-ATPase.     

Coupling of membrane-bound and detergent-solubilized sodium- and potassium-activated ATPase to p-benzoquinone-treated microtiter plates

Membrane-bound and dodecyloctaoxyethyleneglycol monoether-solubilized Na,K-ATPases from pig kidney were covalently attached to microtiter plate wells pretreated with p-benzoquinone (plus collodion for some plates). The immobilized enzymes were detected with the mouse monoclonal antibody (named 38) specific to Na,K-ATPase and a perioxidase-conjugated rabbit IgG anti-mouse IgG. When the two Na,K-ATPase preparations were applied to each well at the same protein concentration, the color intensity of the peroxidase reaction for determination of antibody was two to three times stronger with the solubilized enzyme than with the membrane-bound enzyme. Similar titer values were obtained from the graphical analysis of titration curves of both enzymes. Red cell membrane proteins as well as Na,K-ATPase were covalently attached to the plastic. p-Benzoquinone should be generally useful for coupling membrane proteins, even in detergent solutions, to microtiter plate wells.     

Differential sensitivity of metaphase to diamide and ouabain in HeLa cells

We have examined the effects of diamide, an oxidizer of glutathione, on the progress of HeLa cells through the cell cycle. At concentrations which do not significantly alter generation time, anaphase or cytokinesis, diamide causes a two-fold increase in the duration of metaphase. At 3 X 10(-8) M, ouabain also prolongs metaphase without effect on anaphase or cytokinesis, though with a different time course. The data suggest that the metaphase stage of mitosis is particularly sensitive to alterations in both sulfhydryl groups and Na+ levels but that the effects of diamide are probably not primarily due to the oxidation of sulfhydryl groups of the Na+/K+-ATPase.     

Variant HeLa cells selected for their resistance to ouabain

The cardiac glycoside, ouabain, normally kills HeLa cells at concentrations of about 10⁻⁷ m or greater. By treating a population of HeLa cells with increasingly higher concentrations of the drug, a variant population was obtained of HeLa cells capable of growing in medium containing 10⁻⁴ M ouabain. Inhibition of volume regulation of cells subjected to hypotonic shock was used as a measure of inhibition of active transport of Na across the plasma membrane. In that way dose-response curves for the rapid effects of ouabain and other inhibitors of active Na transport were obtained with both the original, ouabain-sensitive (OS) and the variant, ouabain-resistant (OR) cells. Three other cardiac glycosides (digoxin, digitoxin and hellebrin) and two aglycones (digitoxigenin and strophanthidjn) were found to be equally as effective as ouabain in inhibiting volume regulation of the OS cells; the concentration which produced half-maximum inhibition, I(max/2), was about 6 × 10⁻⁷ M in each case. Similar inhibition of the OR population by ouabain was observed only when the concentration exceeded 10⁻⁴ m [I(max/2)∼2.5 × 10⁻⁴ m], and the other steroid compounds had no effect on the variant cells at the highest concentrations tested (∼2 × 10⁻⁵ m). OR and OS cells differed also in their sensitivities to the cardioactive erythrophleum alkaloid, coumingine; I(max/2) for OS and OR cells was 5 × 10⁻⁸ m and 6 × 10⁻⁷ M, respectively. These results, in addition to results of ouabain binding experiments and measurements of the rates of reversal of inhibition of volume regulation, suggest that a major reason for the differential sensitivities of the two phenotypes to these drugs is different affinities of their sodium pumps for inhibitors of active transport.     

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.     

The rate-limiting step in the actomyosin adenosinetriphosphatase cycle

We have previously shown that myosin does not have to detach from actin during each cycle of ATP hydrolysis. In the present study, using the A-1 isoenzyme of myosin subfragment 1, we have investigated the nature of the rate-limiting steps in the ATPase cycle. Our results show that, at 15 degrees C, at very low ionic strength, KATPase determined from the double-reciprocal plot of ATPase activity vs. actin concentration is more than 6-fold stronger than KBINDING determined by directly measuring the binding of A-1 myosin subfragment 1 to actin during steady-state ATP hydrolysis. Computer modeling shows that this large difference between KATPase and KBINDING is not compatible with Pi release being the rate-limiting step in the ATPase cycle. If Pi release is not rate limiting, it is possible that the ATP hydrolysis step, itself, is rate limiting. However, this predicts that, at high actin concentration, the value of the initial Pi burst should be close to zero. Therefore, we measured the magnitude of the initial Pi burst in the presence of actin, using both direct measurement and measurement of relative fluorescence magnitude. Our results suggest that the magnitude of the initial Pi burst in the presence of actin is considerably higher than would be expected if the ATP hydrolysis step were the rate-limiting step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of sodium- and potassium-activated adenosine triphosphatase. Time course of intermediary steps examined by computer simulation of transient kinetics.

In order to learn whether the kinetics of transient phosphorylation of sodium plus potassium ion transport adenosine triphosphatase was compatible with the hydrolysis of ATP, computer simulation of experimental data was studied. The enzyme mechanism was described in terms of first order and pseudo-first order reactions. The resulting system of linear first order differential equations was solved by a Runge-Kutta method. Phosphorylation kinetics was studied by means of a rapid mixing apparatus at 21 degrees in the presence of 100 micron ATP, 3 mM MgCl2, 120 mM NaCl, and 10 mM KCl. Computer simulation gave a close fit to experimental data with a model of the reaction mechanism which included a sequence of two dephospho forms and two phospho forms of the enzyme. With this model, rate constants obtained by computer simulation were in agreement with constants which had been determined in separate phosphorylation and dephosphorylation experiments. Within experimental limits, the net flux of reaction in each partial step was compatible with the (Na+,K+)-stimulated hydrolysis of ATP (about 324 and 300 nmol-mg-1-min-1, respectively).     

Directional immobilization of sodium- and potassium-activated ATPase to expose its cytoplasmic part to the liquid phase on microtiter plates by wheat germ agglutinin

A convenient method for highly efficient and directional immobilization of intact sodium- and potassium-activated ATPase (Na,K-ATPase) using wheat germ agglutinin linked on microtiter plates was developed. Wheat germ agglutinin, which bound tightly to the beta-subunit of Na,K-ATPase and had no effect on the Na,K-ATPase activity, the potassium-activated p-nitrophenylphosphatase activity, or the inhibitory action of ouabain, was covalently linked to microtiter plates and used as an immobilizer of the enzyme. The amount of Na,K-ATPase coupled to microtiter plates in this immobilizing system was more than 10-fold greater than that used in the direct immobilizing system (O. Urayama, M. Nakao, H. Nagamune, and H. Sugiyama, (1984) Anal. Biochem. 141, 194-198). Also in this system, the cytoplasmic domain of Na,K-ATPase was exposed to the liquid phase. This technique was useful for investigating the reactivities of monoclonal antibody specific for the cytoplasmic domain of the enzyme. Moreover, because this technique was used successfully in the immobilization of periodic acid--Schiff positive staining glycoprotein 1 prepared from human erythrocytes and human alpha 2-macroglobulin, the technique should also be useful for other membrane or secreted proteins that possess N-linked sugar chains containing bisecting N-acetylglucosamine or a high amount of sialic acid.     

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.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Purification of human tonsil plasma cells: pre-enrichment step by immunomagnetic selection of CD31(+) cells

BACKGROUND: The advancement of knowledge about the biology of human normal plasma cells (PC) is hampered by their low frequency and difficult isolation. The aim of this study is to design a way of purifying these cells. METHODS: To this end, advantage was taken of the fact that human tonsil PC expressed surface CD31 at higher levels than the rest of tonsil B cells. RESULTS: The immunomagnetic selection of CD31(+) cells from tonsil B cells increased by a factor of 12 the proportion of PC, determined as CD38(high) cells. This method recovered half of the initial number of PC, and did not alter the PC functions, because IgG secretion was similar in control B cell cultures as well as in cultures of B cells obtained at successive steps of the selection procedure. In addition, CD38(high) cells pre-enriched by this technique were readily isolated by FACS sorting and clearly identified as PC. CONCLUSIONS: Therefore, immunomagnetic pre-enrichment of CD31(+) cells is an efficient method that allows the complete purification of human functional PC.     

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.