The effect of micromolar Ca2+ on the activities of the different Na+/K(+)-ATPase isozymes in the rat myometrium.

In the present work we show the existence of two Na+/K(+)-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca2+ sensitivities. The catalytic subunits (alpha 1, alpha 2) of Na+/K(+)-ATPase were labelled by fluorescein-isothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca2(+)-sensitivities were studied by comparing the kinetics of Ca2+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabain-sensitive part (alpha 2 type) of Na+/K(+)-ATPase enzyme could only be inhibited by micromolar Ca2+. Furthermore, treatment of the microsomal preparation with 1mM N-ethylmaleimide selectively inactivated the high Ca2+ sensitive isoform of myometrial Na+/K(+)-ATPase.     

Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.     

Polarized distribution of Na+, K(+)-ATPase alpha-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Role of the alpha2-isoform of Na+, K(+)-ATPase in the positive inotropic effect of ouabain and marinobufagenin in the rat diaphragm

It was shown that the specific inhibitors of Na+, K(+)-ATPase ouabain and marinobufagenin increased the contraction of an isolated rat diaphragm (positive inotropic effect) by up to approximately 15% in a dose-dependent manner with EC50 = 1.2 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The results indicate the involvement of the ouabain-sensitive alpha 2 isoform of Na+, K(+)-ATPase. The analysis of ouabain-resting membrane potential dose-response relationships in the presence and absence of hyperpolarizing concentration of acetylcholine (100 nM) suggests the existence of two pools of alpha 2 Na+, K(+)-ATPase with different affinities for ouabain. The pool with a higher ouabain affinity is involved in the hyperpolarizing effect of acetylcholine and, presumably, in the positive inotropic effect of ouabain, which might be a mechanism of regulation of muscle efficiency by circulating endogenous inhibitors of Na+, K(+)-ATPase.     

Modulation of Na+-K+-ATPase by calcium ions

The modulatory effects of calcium ions on highly active Na+, K(+)-ATPase from calf brain and pig kidney tissues have been studied. The inhibitory action of Ca2+free on this enzyme depends on the level of ATP (but not AcP). The reduction of pH from 7.4 to 6.0 noticeably increases, but the elevation of pH to 8.0, in its turn, decreases the inhibition of ATP-hydrolyzing activity by calcium. With the increase of K+ concentration (in contrast to Na+) the sensibilization of Na+, K(+)-ATPase to Ca ions is observed. In the presence of potassium ions Mg2+free effectively modifies the inhibitory action of Ca2+free on this enzyme. Ca2+free (0.16-0.4 mM) decreases the sensitivity of Na+, K(+)-ATPase to action of the specific inhibitor ouabain in the presence of ATP. In the presence of AcP (phosphatase reaction) such a change of enzyme sensitivity to ouabain isn't observed. The influence of membranous effects of Ca2+ on the interaction of Na+, K(+)-ATPase with the essential ligands and cardiosteroids is discussed.     

Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin stimulates a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca(2+)-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K(0.5) value for the complex of 15 +/- 1 microg ml(-1), which is unaffected by free [Ca(2+)]. Heparin increases V(max) up to 3-fold while it does not significantly affect the apparent K(m) for free Ca(2+) and for the nucleoside triphosphate substrate. The heparin effect is not additive with that of exogenous calmodulin and heparin is ineffective on a mutant devoid of the N-terminal auto-inhibitory domain (Delta74-ACA8). Altogether, these results indicate that heparin activation is due to partial suppression of the auto-inhibitory function of ACA8 N-terminus. Pull-down assays using heparin-agarose gel show that heparin directly interacts with ACA8. Binding to the heparin-agarose gel occurs also with a peptide reproducing ACA8 sequence (1)M-I(116). Several single-point mutations within ACA8 sequence A56-T63 significantly alter the enzyme response to heparin, suggesting that heparin interaction with this site may be involved in ACA8 activation. These results highlight a new difference between the plant PM Ca(2+)-ATPase and its animal counterpart, which is inhibited by heparin.     

Na+,K(+)-ATPase isozymes in the bovine brain grey matter and brain stem

Active preparations of Na+,K(+)-ATPase containing three types of catalytic isoforms were isolated from the bovine brain to study the structure and function of the sodium pump. Na+,K(+)-ATPase from the brain grey matter was found to have a biphasic kinetics with respect to ouabain inhibition and to consist of a set of isozymes with subunit composition of alpha 1 beta 1, alpha 2 beta m and alpha 3 beta m (where m = 1 and/or 2). The alpha 1 beta 1 form clearly dominated. For the first time, glycosylation of the beta 1-subunit of the alpha 1 beta 1-type isozymes isolated from the kidney and brain was shown to be different. Na+,K(+)-ATPase from the brain stem and axolemma consisted mainly of a mixture of alpha 2 beta 1 and alpha 3 beta 1 isozymes having identical ouabain inhibition constants. In epithelial and arterial smooth muscle cells, where the plasma membrane is divided into functionally and biochemically distinct domains, the polarized distribution of Na+,K(+)-ATPase is maintained through interactions with the membrane cytoskeleton proteins ankyrin and spectrin (Nelson and Hammerton, 1989; Lee et al., 1996). We were the first to show the presence of the cytoskeleton protein tubulin (beta 5-isoform) and glyceraldehyde-3-phosphate dehydrogenase in a high-molecular-weight complex with Na+,K(+)-ATPase in brain stem neuron cells containing alpha 2 beta 1 and alpha 3 beta 1 isozymes. Consequently, the influence of not only subunit composition, but also of glycan and cytoskeleton structures and other plasma membrane-associated proteins on the functional properties of Na+,K(+)-ATPase isozymes is evident.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Ouabain sensitivity of Na+,K+-ATPase in neuronal membranes]

Na+, K(+)-ATPase preparations of the rat and bovine brain and kidney were studied for ouabain sensitivity. Differences in apparent affinities to inhibitor of alpha(+)- and alpha-isozymes of Na+, K(+)-ATPase catalytic subunit were detected only in rat tissues but not in bovine ones. It is concluded that glycoside-sensitive and glycoside-resistant enzymic forms are not fully identical to alpha(+)- and alpha-subunit forms of Na+, K(+)-ATPase.     

The heterogeneity of the Na+, K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

The dose dependence of the Na+, K(+)-ATPase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+, K(+)-ATPase (alpha1-isoform). The specific role of the alpha2- and alpha1- Na+, K(+)-ATPase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Platelet membrane Ca(2+) and Na(+), K(+)-ATPase activity and aggregation in myelodysplastic syndrome

Platelet aggregation was decreased under action of ADP and collagen in patients with myelodysplastic syndrome. The decrease in aggregation started from 3-rd minutes and decreased in 4 and 5 minutes after action of ADP. The study of Ca(2+)-ATPase and Na+, K(+)-ATPase membrane activities showed the decrease in Ca(2+)-ATPase and increase in Na+, K(+)-ATPase activity in the patients with myelodysplastic syndrome.     

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Various properties of the Na+, K(+)-ATPase and the Mg (2+)-ATPase in erythrocytes from normotensive and hypertensive subjects]

In the present work we reported the results of the study of erythrocyte membrane Na+,K(+)-adenosine triphosphatase (ATPase) and Mg(2+)-ATPase in patients with essential hypertension and controls. In the 40 patients with hypertension, a more marked decrease of Na+, K(+)-ATPase was observed. The behavior of the enzyme at Mg2+ activation, ouabain inhibition and the response to different temperature suggest the possibility of differences between the two groups. The normal erythrocyte Mg(2+)-ATPase activity in two groups suggest also the possible role of ratio Na+, K(+)-ATPase/Mg(2+)-ATPase in the study of essential hypertension. However the relevance of magnesium and Mg(2+)-ATPase to the pathogenesis of essential hypertension remains unclear but merits further study. On the basis of these considerations the aim of the present study was to identify, in a kinetic approach, the presence of different abnormalities of Na+ transport and Na+, K(+)-ATPase in erythrocytes from patients with essential hypertension. Much evidence has supported the hypothesis that essential hypertension is a heterogeneous disease in the pathophysiological mechanisms as well as in its clinical and therapeutical consideration.     

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.