Allosteric property of the (Na+K)-ATPase β1 subunit

(Na⁺+K⁺)-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na⁺ and K⁺ ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β₁ subunit can generate a significant impact on the catalytic function of NKA. By using a β₁ subunit-specific monoclonal antibody JY2948, we found that the JY2948–β₁ subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na⁺ and K⁺ ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β₁ subunit and uncovers the latent allosteric property of the β₁ subunit, which remotely controls the NKA catalytic function.     

Tryptic digest of the α subunit of lamb kidney (Na+ + K+)-ATPase

The M_r ≈ 100 000 α subunit was prepared from highly purified lamb kidney (Na⁺+ K⁺)-ATPase. Its N-terminal sequence is Gly-Arg-Asx-Lys-Tyr-Glu. The α subunit was S-carboxymethylated, succinylated, and cleaved at its 40 arginine residues with trypsin. Four major, well-differentiated peptide fractions (A to D) were obtained by chromatography of the digest on a Sephadex G-50 column. Fraction A eluted at the void volume of the column and contained aggregated, very hydrophobic peptides, possibly from regions of α that are buried within the membrane lipid bilayer in the native enzyme. Fractions B to D, which together accounted for about 75% of the total protein, contained water-soluble peptides. To test the feasibility of using antibodies to identify and purify specific peptides of α subunit, studies were carried out using antibodies to native (Na⁺+ K⁺)-ATPase. Carboxymethylation and succinylation did not significantly decrease total antibody binding to α subunit, although the affinity of the anti-(Na⁺ + K⁺)-ATPase antibodies for α subunit was reduced by about 50%. The tryptic peptides of a subunit also retain significant immunochemical reactivity. Fractions A, B and C (but not D) of the digest all bind antibodies. To characterize further the tryptic digest, 16 peptides from fraction D were isolated and sequence studies on these were carried out.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

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.     

Evidence for essential disulfide bonds in the β-subunit of (Na+ + K+)-ATPase

$\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from dog kidney lost its activity when heated at 55°C in the presence of 0.3 M 2-mercaptoethanol. Either heat treatment alone or addition of reducing agent at around 25°C caused little inactivation. One disulfide bond per protomer (mol. wt. 146000) was reduced in the inactivated sample but in active samples no reduction occurred. Neither K⁺-dependent phosphatase activity nor phosphoenzyme formation in the presence of Na⁺ was detected in the inactivated sample, suggesting that the disulfide bond was essential for the catalytic cycle of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. This essential disulfide bond belonged to the β-subunit, the glycoprotein component of the enzyme, indicating that the β-subunit may be an integral component of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ system.     

Serine496 of β2 subunit of L-type Ca2+ channel participates in molecular crosstalk between activation of (Na++K+)-ATPase and the channel

Activation of (Na++K+)-ATPase (NKA) regulates cardiac L-type Ca2+ channel (LTCC) function through molecular crosstalk. The mechanism underlying NKA-LTCC crosstalk remains poorly understood. We have previously shown that activation of NKA leads to phosphorylation of LTCC α1 Ser1928. Here we investigated whether LTCC β2 subunit is modulated by NKA activation and found that LTCC β2 Ser496 is phosphorylated in response to activation of NKA. Src inhibitor PP1 and Erk1/2 inhibitor PD98059 abolish LTCC β2 Ser496 phosphorylation, suggesting that NKA-mediated β2 Ser496 phosphorylation is dependent of Src/Erk1/2 signaling pathway. Protein kinase G (PKG) inhibitor KT5823 failed to inhibit the phosphorylation of β2 Ser496, indicating that the NKA-LTCC crosstalk is independent of PKG activity. The results of nifedipine sensitive 45Ca influx experiments suggest that phosphorylation of β2 Ser496 may play a key down-regulation role in attenuating the accelerated activity of α1 subunit of the channel. Ouabain does not cause a phosphorylation on β2 Ser496, indicating a fundamental difference between activation and inhibition of NKA-mediated biological processes. This study provides the first evidence to demonstrate that LTCC β2 subunit is coupled with the movement of signals in the mechanism of activation of NKA-mediated crosstalk with LTCC.     

Serine of β2 subunit of L-type Ca channel participates in molecular crosstalk between activation of (Na+K)-ATPase and the channel

Activation of (Na⁺+K⁺)-ATPase (NKA) regulates cardiac L-type Ca²⁺ channel (LTCC) function through molecular crosstalk. The mechanism underlying NKA-LTCC crosstalk remains poorly understood. We have previously shown that activation of NKA leads to phosphorylation of LTCC α₁ Ser¹⁹²⁸. Here we investigated whether LTCC β₂ subunit is modulated by NKA activation and found that LTCC β₂ Ser⁴⁹⁶ is phosphorylated in response to activation of NKA. Src inhibitor PP1 and Erk1/2 inhibitor PD98059 abolish LTCC β₂ Ser⁴⁹⁶ phosphorylation, suggesting that NKA-mediated β₂ Ser⁴⁹⁶ phosphorylation is dependent of Src/Erk1/2 signaling pathway. Protein kinase G (PKG) inhibitor KT5823 failed to inhibit the phosphorylation of β₂ Ser⁴⁹⁶, indicating that the NKA-LTCC crosstalk is independent of PKG activity. The results of nifedipine sensitive ⁴⁵Ca influx experiments suggest that phosphorylation of β₂ Ser⁴⁹⁶ may play a key down-regulation role in attenuating the accelerated activity of α₁ subunit of the channel. Ouabain does not cause a phosphorylation on β₂ Ser⁴⁹⁶, indicating a fundamental difference between activation and inhibition of NKA-mediated biological processes. This study provides the first evidence to demonstrate that LTCC β₂ subunit is coupled with the movement of signals in the mechanism of activation of NKA-mediated crosstalk with LTCC.     

Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

The Na⁺/K⁺-ATPase mediates electrogenic transport by exporting three Na⁺ ions in exchange for two K⁺ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb⁺ ions in the crystal structures of the Na⁺/K⁺-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na⁺ or K⁺ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na⁺ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na⁺/K⁺-ATPase α₂ subunit decreases the affinity for extracellular and intracellular Na⁺, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na⁺ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K⁺, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na⁺ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na⁺/Na⁺ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na⁺ release/binding mechanism. In analogy to the mechanism proposed for the H⁺ leak currents of the wild-type Na⁺/K⁺-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na⁺ occlusion reaction, thus destabilizing the Na⁺-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na⁺/K⁺-ATPase α subunit represents a structural and functional relay between Na⁺ binding site III and the intracellular cation occlusion gate.     

Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na⁺, K⁺)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg⁻¹ H₂O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg⁻¹ H₂O). Hemolymph [Na⁺] (323.0 ± 2.5 mmol L⁻¹) and [Mg²⁺] (34.6 ± 1.0 mmol L⁻¹) are hypo-regulated while [Ca²⁺] (22.5 ± 0.7 mmol L⁻¹) is hyper-regulated; [K⁺] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L⁻¹) but hypo-regulated (6.2 ± 0.7 mmol L⁻¹) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na⁺, K⁺)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg⁻¹; K_0.5 = 7.07 ± 0.01 μmol L⁻¹) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg⁻¹; K_0.5 = 0.11 ± 0.3 mmol L⁻¹), both obeying cooperative kinetics, were disclosed. Modulation of (Na⁺, K⁺)-ATPase activity by Mg²⁺, K⁺ and NH₄⁺ also exhibits site-site interactions, but modulation by Na⁺ shows Michaelis-Menten kinetics. (Na⁺, K⁺)-ATPase activity is synergistically stimulated up to 45% by NH₄⁺ plus K⁺. Enzyme catalytic efficiency for variable [K⁺] and fixed [NH₄⁺] is 10-fold greater than for variable [NH₄⁺] and fixed [K⁺]. Ouabain inhibited ≈80% of total ATPase activity (K_I = 464.7 ± 23.2 μmol L⁻¹), suggesting that ATPases other than (Na⁺, K⁺)-ATPase are present. While (Na⁺, K⁺)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min⁻¹ mg⁻¹) and 45‰-acclimated crabs (around 154 nmol Pi min⁻¹ mg⁻¹), ATP affinity decreases 110-fold and Na⁺ and K⁺ affinities increase 2-3-fold in 45‰-acclimated crabs.     

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.     

Stiffness of γ subunit of F1-ATPase

F₁-ATPase is a molecular motor in which the γ subunit rotates inside the α₃β₃ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F₁-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F₁ and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F₁ from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α₃β₃ ring, and the complex of the external part of the γ subunit and the α₃β₃ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F_o and F₁-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.     

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.     

FXYD2, a γ subunit of Na+,K+-ATPase,maintains persistent mechanical allodynia induced by inflammation

Na⁺,K⁺-ATPase (NKA) is required to generate the resting membrane potential in neurons. Nociceptive afferent neurons express not only the α and β subunits of NKA but also the γ subunit FXYD2. However, the neural function of FXYD2 is unknown. The present study shows that FXYD2 in nociceptive neurons is necessary for maintaining the mechanical allodynia induced by peripheral inflammation. FXYD2 interacted with α1NKA and negatively regulated the NKA activity, depolarizing the membrane potential of nociceptive neurons. Mechanical allodynia initiated in FXYD2-deficient mice was abolished 4 days after inflammation, whereas it persisted for at least 3 weeks in wild-type mice. Importantly, the FXYD2/α1NKA interaction gradually increased after inflammation and peaked on day 4 post inflammation, resulting in reduction of NKA activity, depolarization of neuron membrane and facilitation of excitatory afferent neurotransmission. Thus, the increased FXYD2 activity may be a fundamental mechanism underlying the persistent hypersensitivity to pain induced by inflammation.     

The Na(+)-translocating F₁F₀-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9M Na(+), pH(50°C) 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F(1) portion and 3-subunit-F(O) portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na(+) (5mM); hydrolysis activity was inhibited by higher Na(+) concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH(50°C) of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg(2+) and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na(+)-ion gradient that was sensitive to the Na(+)-ionophore monensin. Analysis of the atp operon showed the presence of the Na(+)-binding motif in the c subunit (Q(33), E(66), T(67), T(68), Y(71)), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F(1)F(O)-ATPase of N. thermophilus is a Na(+)-translocating ATPase used primarily for expelling cytoplasmic Na(+) that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na(+)-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na(+) needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.     

Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄ ⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K _M = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺)-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K _0.5 = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄ ⁺ had no modulatory effect. The affinity for Na⁺ (K _0.5 = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄ ⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄ ⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.     

Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the α- and β-subunits of (Na⁺ + K⁺)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[¹²⁵I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na⁺ + K⁺)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na⁺ + K⁺)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of ¹²⁵I-labeling within the α-subunit. The preservation of (Na⁺ + K⁺)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E₁- and E₂-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.