Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Interaction of alcohol and protein deficiency on rat brain synaptosomal (Na+−K+)-ATPase

Administration of low amounts of ethanol for a prolonged period increases rat brain synaptosomal (Na⁺–K⁺)-ATPase activity, the increase being less in the protein deficient rats. The adaptive mechanism to offset the stress imposed by the continued presence of ethanol seems to be depressed by low plane of nutrition. In vivo and in vitro effects of ethanol on (Na⁺–K⁺)ATPase seems to be different.     

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+−Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve

The ATP hydrolysis dependent Na⁺-Na⁺ exchange of reconstituted shark (Na⁺ + K⁺)-ATPase is electrogenic with a transport stoichiometry as for the Na⁺-K⁺ exchange, suggesting that translocation of extracellular Na⁺ is taking place via the same route as extracellular K⁺. The preparation thus offers an opportunity to compare the sided action of Na⁺ and of K⁺ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K⁺. With Na⁺ but no K⁺ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K⁺ in concentrations of 50 μM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K⁺ also gives biphasic ATP-activation curves, however, only when the K⁺ concentration is 50 mM or higher (Na⁺ + K⁺ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E₂(Na₂⁺) and E₂(K₂⁺), respectively, and in which the dephosphorylation of E₂-P is rate limiting in the presence of Na⁺ (but no K⁺) extracellular, whereas in the presence of extracellular K⁺ it is the deocclusion of E₂(K₂⁺) which is rate limiting.     

The α1 Na+−K+ pump of the Dahl salt-sensitive rat exhibits altered Na+ modulation of K+ transport in red blood cells

The properties of the α1 Na⁺-K⁺ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( _i ) and external ( _o ) Na⁺ and K⁺ concentrations. Kinetic parameters of several modes of operation, i.e., Na⁺/ K⁺, K⁺/K⁺, Na⁺/Na⁺ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na⁺ + 5 mm K⁺, ouabain-sensitive K⁺ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na⁺ efflux and Na _i were similar in both strains. Thus, the coupling ratio between unidirectional Na⁺∶K⁺ fluxes was significantly higher in DS than in DR cells at similar RBC Na⁺ content. In the presence of 140 mm Na _o , activation of ouabain-sensitive K⁺ influx by K _o had a lower K _m and V _max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K _m 0.74 ± 0.09 mm; V _max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K _m 2.31 ± 0.16 mm; v _max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na _o removal. The activation of ouabain-sensitive K⁺ influx by Na _i had significantly lower V _max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K _m. These data suggest that the low K⁺ influx in DS cells is caused by a defect in modulation by Na _o and Na _i . Na⁺ efflux showed no differences in Na _i activation or trans effects by Na _o and K _o , thus accounting for the different Na⁺∶K⁺ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na⁺ and K⁺ fluxes, where the net ouabain-sensitive Na⁺ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K⁺ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na⁺ influx and K⁺ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na⁺ influx by K _o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K _o could activate higher K⁺ influx in DR pumps without a complete inhibition of ouabain-sensitive Na⁺ influx. This behavior is consistent with K _o interaction with distinct Na⁺ and K⁺ transport sites. In addition, the inhibition of K⁺ efflux by Na, was different between Dahl strains. Ouabain-sensitive K⁺ efflux at Na _i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K₅₀ for Na _i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na⁺ modulation of K⁺ transport is altered at both sides of the membrane. The dissociation of Na⁺ modulatory sites of K⁺ transport from Na⁺ transport sites observed in RBCs of Dahl strains suggests that K⁺ transport by the Na⁺-K⁺ pump is controlled by Na⁺ allosteric sites different from the Na⁺ transport sites. The alterations in K⁺ transport may be related to the amino acid substitution (Leu/Gln₂₇₆) reported for the cDNA of the α1 subunit of the Na⁺-K⁺ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na⁺-K⁺ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990.     

Stimulation of (Na+K+)-ATPase of rat liver plasma membrane by amino acids

The effect of twelve l-amino acids on the activity of liver plasma membrane (Na⁺K⁺)-ATPase has been tested. Histidine and arginine significantly enhanced the activity. The activtion by histidine showed saturation kinetics with an apparent K_a of about 8 mM, and was evident over a wide range of Na⁺ concentrations. The same amino acid did not significantly affect the Mg²⁺-dependent ATPase activity.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by trypsin digestion

Summary To distinguish ligand-induced structural states of the (Na⁺–K⁺)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na⁺, K⁺, Mg⁺⁺ and ATP. It was found that first the large and then the small polypeptide chain of the (Na⁺–K⁺)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na⁺–K⁺)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na⁺–K⁺)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released.It was found that the combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ present during trypsin digestion influenced the time course and degree of degradation of the (Na⁺–K⁺)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced.In the absence of ATP the time course of trypsin digestion of the (Na⁺–K⁺)-ATPase was the same, whether Na⁺ or K⁺ was present. With low ATP concentrations (e.g., 0.1mm), however, binding of Na⁺ or K⁺ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g., 10mm) was present, Na⁺ and K⁺ also influenced the degradation pattern of the (Na⁺–K⁺)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na⁺ and K⁺ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ if the large chain was intact when the ligands were added to the enzyme.The described results demonstrate structural alterations of the (Na⁺–K⁺)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na⁺–K⁺)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na⁺–K⁺)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na⁺–K⁺)-ATPase is regulated by binding of Na⁺ or K⁺ to the enzyme-ATP complex. The effects of Na⁺ and K⁺ on the (Na⁺–K⁺)-ATPase structure are modulated by the ATP binding to high affinity and to low affinity ATP binding sites.     

Variability of the K+−Na+ discrimination of beauvericin in mitochondrial membranes

In intact mitochondria supplemented with succinate or -hydroxybutyrate, the rates of oxygen consumption induced by beauvericin followed the ionic selectivity pattern: Na⁺>Rb⁺, Cs⁺, K⁺, Li⁺.When the respiratory substrate is glutamate plus malate in the absence of phosphate, the selectivity pattern is: K⁺>Rb⁺>Cs⁺>Li⁺>Na⁺.When the media are supplemented with phosphate, the Na⁺/K⁺ discrimination of beauvericin is considerably modified with all the respiratory substrates, being K⁺>Na⁺ with succinate and Na⁺>K⁺ with glutamate plus malate, whereas no significant ionic selectivity differences were obtained with -hydroxybutyrate.The respiratory control induced by oligomycin in submitochondrial particles is released by beauvericin only in the presence of a nigericin-like carboxylic antibiotic and an alkali metal cation, being far more effective in K⁺ than in Na⁺.This selectivity is maintained regardless of whether NADH or succinate is used as a respiratory substrate.Release of respiratory control can also be obtained with a combination of beauvericin and NH₄Cl.This information indicates that the ionic selectivity pattern obtained with beauvericin in mitochondrial membranes is an intrinsic property of the antibiotic which, however, can be significantly modified by factors such as the nature of the translocatable substrate anion or other anionic species, as well as the possible operation of a Na⁺/H⁺ antiporter existent in the membrane.     

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

Capsazepine (CPZ) inhibits Na⁺,K⁺-ATPase-mediated K⁺-dependent ATP hydrolysis with no effect on Na⁺-ATPase activity. In this study we have investigated the functional effects of CPZ on Na⁺,K⁺-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na⁺,K⁺-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na⁺,K⁺-ATPase current in the presence of extracellular K⁺. In contrast, CPZ stimulated pump current in the absence of extracellular K⁺. Similar conclusions were attained using HEK293 cells loaded with the Na⁺ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na⁺,K⁺-ATPase indicated that CPZ stabilizes ion interaction with the K⁺ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na⁺ congener at the Na⁺ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na⁺,K⁺-ATPase. CPZ increased oxonol VI fluorescence in the absence of K⁺, reflecting increased Na⁺ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K⁺, likely indicating opening of an intracellular pathway selective for K⁺. As revealed by the recent crystal structure of the E₁.AlF₄ ^-.ADP.3Na⁺ form of the pig kidney Na⁺,K⁺-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K⁺ sites in Na⁺,K⁺-ATPase.     

Role of the Na+,K+-ATPase α2 isoform in the positive inotropic effect of ouabain and marinobufagenin in the rat diaphragm

It was found that ouabain and marinobufagenin, specific inhibitors of Na⁺,K⁺-ATPase, increased the contraction of the isolated rat diaphragm by ～15% (positive inotropic effect) at EC₅₀ = 1.2 ± 0.3 nM and 0.3 ± 0.1 nM, respectively, which was indicative of the participation of the ouabain-sensitive Na⁺,K⁺-ATPase α2 isoform. Analysis of the dose-response curves for the effect of ouabain on the resting membrane potential of muscle fibers in the absence and in the presence of 100 nM acetylcholine (hyperpolarizing the membrane) showed the presence of two pools of Na⁺,K⁺-ATPase α2 that differed in affinity for ouabain. Only the high-affinity pool (IC₅₀ ～ 9 nM) mediates the hyperpolarizing effect of nanomolar concentrations of acetylcholine. Most likely, it is this pool of that is involved in the positive inotropic effect of ouabain, which can be a mechanism of regulation of the muscle function by circulating endogenous inhibitors of Na⁺,K⁺-ATPase.     

Inhibition of Na+, K+-ATPase activity by δ-aminolevulinic acid

-Aminolaevulinic acid (ALA) has been shown to be toxic to cultured neurons and glia at concentrations as low as 10 M. In an attempt to elucidate the mechanism of toxicity, the effects of ALA on membrane ATPase activity were investigated. Exposure of neuron cultures to 1 mM ALA for 7 days caused a substantial decrease in both Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities. At lower concentrations, ALA affected only the Na⁺, K⁺-component. ALA appeared to act directly, inhibiting Na⁺, K⁺-ATPase activity in rat brain cortex membrane preparations at 10 M Although this effect was slight, it may well represent the mechanism of action of ALA, since ouabain, a potent inhibitor of Na⁺, K⁺-ATPase activity, proved to be more toxic to cultured neurons than ALA. Furthermore, cardiac glycoside overdosage causes neurological disturbances which are very similar to those observed in the acute attack of porphyria.     

The Polarized Distribution of Na+,K+-ATPase: Role of the Interaction between β Subunits

The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na⁺,K⁺-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na⁺,K⁺-ATPase polarity depends on interactions between the β subunits of Na⁺,K⁺-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these β subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

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.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Summary Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na⁺–Ca²⁺ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na⁺–Ca²⁺ exchange. The treatment with dithiothreitol resulted in a decrease inV _max values but did not alter theK _m for Ca²⁺ for the Na²⁺–Ca²⁺ exchange reaction. If Na⁺ replaced K⁺ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na⁺–Ca²⁺ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N-ethylmaleimide, were capable of altering Na⁺–Ca²⁺ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na⁺–Ca²⁺ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na⁺–Ca²⁺ exchange, depending on whether Na⁺ or K⁺ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfurcontaining residues interact with a Na⁺-binding site for Na⁺–Ca²⁺ exchange in cardiac sarcolemmal vesicles.     

Effect of retinol deficiency and curcumin or turmeric feeding on brain Na+−K+ adenosine triphosphatase activity

The effect of retinol deficiency and curcumin and turmeric feeding on brain microsomal Na⁺-K⁺ ATPase activity was investigated. The brain Na⁺–K⁺ ATPase activity registered an increase of 148.5% as compared to the control group. Upon treating retinol deficient rats with curcumin or turmeric, the abnormally elevated activity showed a decrease of 36.9 and 47.1%, respectively, when compared to the retinol deficient group. An increase in V_max by 67% and K_m by 66% for ATP was observed in the retinol deficient group. Curcumin or turmeric fed retinol-deficient groups reduced the V_max by 25 and 33%, while K_m was reduced by 25 and 31%, respectively, compared to the retinol deficient group. Arrhenius plot of Na⁺–K⁺ ATPase showed a typical bi-phasic pattern in all the groups. Cholesterol: Phospholipid ratio showed a decrease in the retinol-deficient group by 67.8%, which showed a marked increase in curcumin or turmeric treated groups. Detergents could increase the Na⁺–K⁺ ATPase activity more in the control group than in the retinol deficient groups. Curcumin or turmeric improved the detergent action on the enzyme. Subsequent freezing and thawing over a period of 30 min decreased the enzyme activity by 22.8% in the retinol deficient group compared to 15.9% decrease in the control group. Curcumin or turmeric treated groups showed a decrease in the enzyme activity by 22.0 and 19.2%, respectively, when compared to the zero time in each group. In the presence of concanavalin-A (Con-A) there was only 52.4% stimulation in the enzyme activity in retinol deficient groups, compared to 108.0% in the control group. Curcumin or turmeric treated retinol-deficient groups showed a stimulation in the presence of con-A by 70 and 99.5%, respectively.