Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets

We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

Effect of polyamines on Mg(2+)-dependent ATP-hydrolase activity in plasmatic membrane of myometrium cells

Influence of aliphatic polyamines of spermine and spermidine on the enzymatic activity of the ouabain-sensitive Na+,K(+)-ATPase and the ouabain-resistant basal Mg(2+)-ATPase (specific activity--10.6 +/- 0.9 and 18.1 +/- 1.2 microM P(i)/hour on 1 mg of protein accordingly, n = 7) has been studied in the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that the polyamine spermine in concentration of 1 and 10 mM activated the Na+,K(+)-ATPase by 54 and 64% on the average relative to control value. Spermidine also stimulated the Na+,K(+)-ATPase activity, however it did it less efficiently than spermine: by 8 and 20% on the average at concentration of 1 and 10 mM, accordingly. Similarly, polyamines had affect on the basal Mg(2+)-ATPase: spermine in concentration of 1 and 10 mM activated it by 26 and 39% relative to control value; spermidine in concentration of 1 and 10 mM activated it by 10 and 32% relative to control. The magnitudes of the apparent activation constant K(a) of spermine were 0.35 +/- 0.07 and 0.10 +/- 0.02 mM for Na+,K(+)-ATPase and basal Mg(2+)-ATPase, accordingly (M +/- m, n = 5). It is supposed, that the obtained experimental data can be useful in the further research of the membrane mechanisms underlying of the cationic exchange in the smooth muscles, in particular, when investigating the role of the plasmatic membrane in providing electromechanical coupling in them, and also in regulation of ionic homeostasis in the smooth muscle cells.     

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.     

Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes

The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 > alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.     

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)- ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal- lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)- dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B- cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Two novel peripheral membrane proteins, pasin 1 and pasin 2, associated with Na+,K(+)-ATPase in various cells and tissues

Purification of pig kidney Na+,K(+)-ATPase at low concentrations of SDS (0.5%) allowed copurification of several peripheral membrane proteins. Some of these associated proteins were identified as components of the membrane cytoskeleton. Here we describe two novel globular proteins of of Mr 77,000 (pasin 1) and Mr 73,000 (pasin 2) which copurify and coimmunoprecipitate with Na+,K(+)-ATPase and can be stripped off Na+,K(+)-ATPase microsomes by 1 M KCl. Pasin 1 and pasin 2 were detected by immunoblot analysis in various cells and tissues including erythrocytes and platelets. Immunostaining revealed colocalization of pasin 1 and Na+,K(+)-ATPase along the basolateral cell surface of epithelial cells of kidney tubules and parotid striated ducts (titers of pasin 2 antibodies were too weak for immunocytochemistry). In erythrocytes, pasin 1 and pasin 2 are minor components bound to the cytoplasmic surface of the plasma membrane. Pasin 1 showed the same electrophoretic mobility as protein 4.1b. However, both proteins have different isoelectric points (pasin 1, pI 6; protein 4.1, pI 7), different chymotryptic fragments, and are immunologically unrelated. Short pieces of sequence obtained from pasin 1 and pasin 2 were not found in any other known protein sequence. The occurrence of pasin 1 and pasin 2 in diverse cells and tissues and their association with Na+,K(+)-ATPase suggests a general role of these proteins in Na+,K(+)- ATPase function.     

The energy transduction mechanism is different among P-type ion-transporting ATPases. Acetyl phosphate causes uncoupling between hydrolysis and ion transport in H+,K(+)-ATPase.

H+,K(+)-ATPase, Na+,K(+)-ATPase, and Ca(2+)-ATPase belong to the P-type ATPase group. Their molecular mechanisms of energy transduction have been thought to be similar until now. Ca(2+)-ATPase and Na+,K(+)-ATPase are phosphorylated from both ATP and acetyl phosphate (ACP) and dephosphorylated, resulting in active ion transport. However, we found that H+,K(+)-ATPase did not transport proton nor K+ when ACP was used as a substrate, resulting in uncoupling between energy and ion transport. ACP bound competitively to the ATP-binding site of H+,K(+)-ATPase. The hydrolysis of ACP by H+,K(+)-ATPase was stimulated by cytosolic K+, the half-maximal stimulating K+ concentration (K0.5) being 2.5 mM, whereas the hydrolysis of ATP was stimulated by luminal K+, the K0.5 being 0.2 mM. Furthermore, during the phosphorylation from ACP in the absence of K+, the fluorescence intensity of H+,K(+)-ATPase labeled with fluorescein isothiocyanate increased, but those of Na+,K(+)-ATPase and Ca(2+)-ATPase decreased. These results indicate that phosphorylated intermediates of H+,K(+)-ATPase formed from ACP are not rich in energy and that there is a striking difference(s) in the mechanism of energy transduction between H+,K(+)-ATPase and other cation-transporting ATPases.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase

The K0.5 for intracellular sodium of the two forms of (Na+,K+)-ATPase which exist in rat adipocytes (Lytton, J., Lin, J. C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184) has been determined by incubating the cells in the absence of potassium in buffers of varying sodium concentration; these conditions shut off the Na+ pump and allow sodium to equilibrate into the cell. The activity of Na+,K+)-ATPase was then monitored with 86Rb+/K+ pumping which was initiated by adding isotope and KCl to 5 mM, followed by a 3-min uptake period. Atomic absorption and 22Na+ tracer equilibration were used to determine the actual intracellular [Na+] under the different conditions. The K0.5 values thus obtained were 17 mM for alpha and 52 mM for alpha(+). Insulin treatment of rat adipocytes had no effect on the intracellular [Na+] nor on the Vmax of 86Rb+/K+ pumping, but did produce a shift in the sodium ion K0.5 values to 14 mM for alpha (p less than 0.025 versus control) and 33 mM for alpha(+) (p less than 0.005 versus control). This change in affinity can explain the selective stimulation of alpha(+) by insulin under normal incubation conditions. Measurement of the K0.5 for sodium ion of (Na+,K+)-ATPase in membranes isolated from adipocytes revealed only a single component of activation with a low K0.5 of 3.5 or 12 mM in the presence of 10 or 100 mM KCl, respectively. Insulin treatment of the isolated membranes or of the cells prior to membrane separation had no effect on these values.     

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+,K(+)-ATPase activity in human colorectal carcinoma

Na+,K(+)-ATPase activities in macroscopically unchanged mucosa (conditionally normal tissue) and human colorectal carcinoma (mainly low-grade and moderately differentiated adenocarcinomas) have been investigated. Microsomal fractions are similar by dimensions of the membrane fragments detected by photon correlation spectroscopy analysis. The activation optima under digitonin pretreatment of the membrane fractions differ significantly for Na+,K(+)-ATPase and concomitant Mg(2+)-ATPase activity, but are the same in conditionally normal and cancerous tissues. This allows to detect correctly total levels of the Na+,K(+)-ATPase activity in the detergent-pretreated preparations. The moderate decrease of the Na+,K(+)-ATPase activity is revealed in carcinomas. It is concluded that a decrease of activity of the ouabain-sensitive human Na+,K(+)-ATPase is characteristic of colorectal carcinoma.     

Effect of partial hepatectomy on the invertor mechanism of regulation of the Na+,K+-ATPase activity in hepatocytes of rats of various ages]

Age peculiarities of partial hepatectomy effect on the hepatocytes plasma membrane Na+, K(+)-ATPase activity and its insulin-induced stimulation has been studied. It has been shown that partial hepatectomy does not change basal Na+, K(+)-ATPase activity in adult rats. In old partial hepatectomised rats Na+, K(+)-ATPase activity is slightly higher than in control old rats, although this increase is not statistically significant. At the same time, partial hepatectomy acts differently on the insulin-induced Na+, K(+)-ATPase activation in adult and old rats. Insulin activates Na+, K(+)-ATPase at the same extent both in control and partial hepatectomized adult animals. In old hepatectomized rats, but not in old control animals, insulin stimulates Na+, K(+)-ATPase activity as well as. Thus hepatectomy "rejuvenates" old hepatocytes and results in recovery of invertor mechanism of Na+, K(+)-ATPase activation.     

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.     

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.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.