Brain plasmamembrane NA+:,K+-ATPase is inhibited by acetylated tubulin

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the -subunit of Na⁺,K⁺-ATPase. In the present work we show that the interaction of tubulin with Na⁺K⁺-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na⁺K⁺-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Regulation of the Na+/K+-ATPase by insulin: Why and how?

The sodium-potassium ATPase (Na⁺/K⁺-ATPase or Na⁺/K⁺-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na⁺ from cells in exchange for K⁺ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits ( and ) each expressed in several isoforms. Many hormones regulate Na⁺/K⁺ -ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na⁺/K⁺-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na⁺ concentration, altered Na⁺ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na⁺/K⁺-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na⁺/K⁺-ATPase control by insulin in diabetes and related disorders is addressed.     

Isolation and characterization of monoclonal antibodies to (Na + K)-ATPase

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na⁺ + K⁺)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ₁ heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na⁺ + K⁺)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na⁺ + K⁺)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na⁺ + K⁺)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.     

Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase, an effect fully prevented by blockade of neurotensin NT₁ receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na⁺, K⁺-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na⁺, K⁺-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT₁), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Modulations of rabbit erythrocyte ATPase activities induced by in vitro and in vivo exposure to ethanol

Alcohol intake is associated with numerous degenerative disorders, and the detrimental effects of alcohol may be due to its influence on plasma membrane and cellular transport systems. The aim of the present study was to compare in vitro and in vivo effects of ethanol on rabbit erythrocyte ATPase activities and correlate them with ethanol-induced oxidative stress. Age-matched male rabbits were given 5% ethanol in 2% sucrose solution, for 6 weeks ad libitum; control animals were given tap water. Daily intake of ethanol was 5 g/kg body weight; this experimental regimen resulted in an average serum ethanol concentration of 16.77 ± 2.00 mM/l. After this period, blood was collected, serum ethanol concentration was determined and erythrocyte membranes were prepared according to the method of Post et al. Activities of Na⁺/K⁺- and Mg²⁺-ATPases were determined. Thiobarbituric acid-reactive substance (TBARS) assay was used to detect levels of lipid peroxidation, a major indicator of oxidative stress. In vitro ethanol inhibits both Na⁺/K⁺-ATPase and Mg²⁺-ATPase, but Na⁺/K⁺-ATPase is more sensitive to the ethanol-induced inhibition. Increasing concentration of ethanol increased TBARS production, but significant difference was attained only at 5 and 12.5 mM of ethanol. Chronic ethanol consumption induced significant increase in Na⁺/K⁺- and Mg²⁺-ATPase activity, and TBARS production. Our results suggest that increased ATPase activity induced by chronic ethanol consumption is due to oxidative, induced modification of membrane phospholipids and proteins, which are responsible for inhibition of ATPase activity. Increased production of TBARS induced by in vitro exposure to ethanol is not the only factor that influences ATPases activity. Further research is needed to elucidate this relationship.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

The effects of captopril and losartan on erythrocyte membrane Na+/K+- ATPase activity in experimental diabetes mellitus

Diabetes mellitus induces a decrease in sodium potassium-adenosine triphosphatase (Na⁺/K⁺- ATPase) activity in several tissues in the rat and red blood cells (RBC) and nervous tissue in human patients. This decrease in Na⁺/K⁺- ATPase activity is thought to play a role in the development of long term complications of the disease. Angiotensin enzyme inhibitors (ACEi) and angiotensin-II receptor antagonists (ARBs) reduce proteinuria and retard the progression of renal failure in patients with IDDM and diabetic rats. We investigated the effects of captopril and losartan, which are used in the treatment of diabetic nephropathy, on Na⁺/K⁺- ATPase activity. Captopril had an inhibitory effect on red cell plasma membrane Na⁺/K⁺ ATPase activity, but losartan did not. Our study draws attention to the inhibitory effect of captopril on Na⁺/K⁺ ATPase activity. Micro and macro vascular complications are preceeding mortality and morbidity causes in diabetes mellitus. There is a strong relationship between the decrease in Na⁺/K⁺ ATPase activity and hypertension. The non-sulphydryl containing ACEi and ARBs must be the choice of treatment in hypertensive diabetic patients and diabetic nephropathy.     

Regulation of acetylated tubulin/Na+,K+-ATPase interaction by L-glutamate in non-neural cells: involvement of microtubules

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na+,K+-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes with L-glutamate induces dissociation of the acetylated tubulin/Na+,K+-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells. As in the case of astrocytes, the effect of L-glutamate is mediated by its transporters and not by specific receptors. In COS cells, the effect of L-glutamate was reversed by its elimination from culture medium, provided that d-glucose was present. The effect of L-glutamate was not observed when Na+ was replaced by K+ in the incubation medium. The ionophore monensin, in the presence of Na+, had the same effect as L-glutamate. Treatment of cells with taxol prevented the dissociating effect of L-glutamate or monensin. Nocodazole treatment of intact cells or isolated membranes dissociated the acetylated tubulin/Na+,K+-ATPase complex. The dissociating effect of nocodazol does not require Na+. These results indicate a close functional relationship among Na+,K+-ATPase, microtubules, and L-glutamate transporters, and a possible role in cell signaling pathways.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Dual activity of the H1-H2 domain of the (Na(+)+K+)-ATPase

(Na⁺+K⁺)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the α-subunit of the (Na⁺+K⁺)-ATPase is one of the digitalis drug interaction sites of the enzyme. Despite the extensive studies of the inhibitory effect of digitalis on the (Na⁺+K⁺)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na⁺+K⁺)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na⁺+K⁺)-ATPase even in the presence of SSA78. These results suggest that the H1-H2 domain of the (Na⁺+K⁺)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na⁺+K⁺)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis or ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure-function relationships of the (Na⁺+K⁺)-ATPase and the biological processes mediated by the enzyme.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K+-ATPase

Summary Membrane fragments containing the H⁺K^–-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na⁺K⁺-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J. 4:3079–3085). Since H⁺K⁺-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K⁺. By separation of the H⁺ and K⁺ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K⁺ or at low concentrations of K⁺. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K⁺ transport during dephosphorylation is electrogenic, too, in contrast to the Na⁺K⁺-ATPase where the K⁺ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H⁺K⁺-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

Polarized distribution of Na, K-ATPase α-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 α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) 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 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). 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 α1 and α2 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 α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.