Coupling of membrane-bound and detergent-solubilized sodium- and potassium-activated ATPase to p-benzoquinone-treated microtiter plates

Membrane-bound and dodecyloctaoxyethyleneglycol monoether-solubilized Na,K-ATPases from pig kidney were covalently attached to microtiter plate wells pretreated with p-benzoquinone (plus collodion for some plates). The immobilized enzymes were detected with the mouse monoclonal antibody (named 38) specific to Na,K-ATPase and a perioxidase-conjugated rabbit IgG anti-mouse IgG. When the two Na,K-ATPase preparations were applied to each well at the same protein concentration, the color intensity of the peroxidase reaction for determination of antibody was two to three times stronger with the solubilized enzyme than with the membrane-bound enzyme. Similar titer values were obtained from the graphical analysis of titration curves of both enzymes. Red cell membrane proteins as well as Na,K-ATPase were covalently attached to the plastic. p-Benzoquinone should be generally useful for coupling membrane proteins, even in detergent solutions, to microtiter plate wells.     

Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither in the presence nor in the absence of anionic phospholipids did protein kinase phosphorylation of native FXYD10, which relieves the inhibition, affect such cross-linking. Together, this seems to indicate that phosphorylation involves only modest structural rearrangements between the cytoplasmic domain of FXYD10 and the Na,K-ATPase A-domain.     

Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.     

Major organ-specific glycoproteins in isolated brain and kidney membranes identified as Na,K-ATPase subunits by combined glycan-, lectin-, and immunoblotting

In the present work combined glycan-, lectin-, and immunoblotting of isolated brain and kidney membranes shows that the and subunits of Na,K-ATPase are the most abundant glycoproteins. Further,Datura stramonium andGalanthus nivalis agglutinins recognize the Na,K-ATPase subunits in a mutually exclusive manner in membranes from human, rabbit and rat brain or human, rabbit, rat, pig and dog kidney indicating the presence of species-independent organ-typical glycoforms. The glycosylation status is not related to the ouabain-sensitivity. Taken together, the data reveals organ-specific glycoforms of Na,K-ATPase which might have roles for organ identification and recognition.Abbreviations NKA Na,K-ATPase (EC 3.6.1.37) - PAGE polyacrylamide gel electrophoresis in dodecylsulfate - Con-A Concanavalin A - DSA Datura stramonium agglutinin - GNA Galanthus nivalis agglutinin - MAA Maackia amurensis agglutinin - PNA Peanut agglutinin - SNA Sambucus nigra agglutinin - WGA Wheat germ agglutinin Abbreviations used in figures K kidney - B brain - Cr Crude - De Detergent-treated - Fe fetuin - Ct creatinase - I-blot immuno-blot - L-blot lectin-blot     

Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells.

Measurements of internal ion concentrations, amino acid pools, and membrane potential were made across a series of HeLa subclones which are amplified for the genes for the sodium- and potassium-activated ATPase (Na,K-ATPase). These subclones expressed heterogeneous levels of ouabain-binding sites, allowing us to construct a graded amplification series. While [K+]i levels did not vary systematically across the series studied, [Na+]i ranged from 9 to 20 mM as a function of Na,K-ATPase expression. Steady-state accumulation of tetraphenylphosphonium in low versus high potassium was used to measure membrane potential. Values for [Na+]i and the membrane potential were used to calculate the sodium electrochemical potential, which was also found to be a function of Na,K-ATPase expression. Measurements of acid-soluble amino acid pools in cell lysates demonstrated that amino acids which are substrates for sodium-dependent transport systems, or which can potentially exchange through system L for a substrate of a sodium-dependent system, varied as a function of the sodium electrochemical potential. This confirmed our prediction of increased amino acid pool sizes in Na,K-ATPase-amplified lines based on observations of elevated flux through the sodium-independent system L. Finally, we measured lactate production and glycolytic potential in a subset of clones and found that both were reduced in subclones with elevated Na,K-ATPase.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

The role of loop 6/7 in folding and functional performance of Na,K-ATPase

Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human alpha(1)-subunit of Na,K-ATPase. Most mutations reduced Na,K-ATPase activity by less than 50%; however, the mutations R834A, R837A, and R848A reduced Na,K-ATPase activity by 75, 89, and 66%, respectively. Steady-state phosphoenzyme formation from ATP was reduced in mutants R834A, R837A, and R848A, and R837A also had a faster E(2)P --> E(2) dephosphorylation rate compared with the wild-type enzyme. Effects of L6/7 mutations on the phosphorylation domain of the protein were also demonstrated by (18)O exchange, which showed that intrinsic rate constants for P(i) binding and/or reaction with the protein were altered. Although most L6/7 mutations had no effect on the interaction of Na(+) or K(+) with Na,K-ATPase, the E825A, E828A, R834A, and R837A mutations reduced the apparent affinity of the enzyme for both Na(+) and K(+) by 1.5-3-fold. 1-Bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU(3+)), a competitive antagonist of Rb(+) and Na(+) occlusion, was used to test whether charged residues in L6/7 are involved in binding monovalent cations and cation antagonists. Br-TITU(3+) inhibited ouabain binding to wild type Na,K-ATPase with an IC(50) of 30 microM. Ouabain binding to the E825A, E828A, R834A, or R837A mutants was still inhibited by Br-TITU(3+), indicating that Br-TITU(3+) does not bind to charged residues in L6/7. This observation makes it unlikely that L6/7 functions as a cytoplasmic cation binding site in Na,K-ATPase, and together with the effects of L6/7 mutations on phosphate interactions with the enzyme suggests that L6/7 is important in stabilizing the phosphorylation domain and its relationship to the ion binding sites of the protein.     

Use of chemically modified PMMA microspheres for enzyme immobilization

Modified poly(methyl methacrylate) (PMMA) microspheres, about 7microm in diameter, carrying aldehyde groups on their surfaces were synthesized and used as the support for enzyme immobilization. The immobilizing behavior as well as the properties of immobilized enzyme was studied. The amount of bound enzyme can be extended to 76.8mg g(-1) support, which is relatively much higher than other supports. The kinetic investigation derived from three typical models shows that the practical process is more complicated than the ideal condition, with one or more interactions being involved in the immobilization process. The K(m) value is actually larger and V(max) is smaller in the immobilized form than those in the free form. The increased resistance of the immobilized enzyme against the changes of temperature indicates that immobilizing enzyme onto the modified microspheres is useful for enzyme immobilization.     

Identification of a region of the polypeptide chain of Na,K-ATPase α-subunit interacting with 67-kDa melittin-like protein

It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.     

Identification of a potential receptor that couples ion transport to protein kinase activity

In our previous studies, we have demonstrated that the Src-coupled α1 Na/K-ATPase works as a receptor for cardiotonic steroids, such as ouabain, to regulate cellular protein kinase cascades. Here, we explore further the structural determinants of the interaction between the α1 Na/K-ATPase and Src and demonstrate that the Src-coupled α1 Na/K-ATPase allows the cell to decode the transmembrane transport activity of the Na/K-ATPase to turn on/off protein kinases. The α1 Na/K-ATPase undergoes E1/E2 conformational transition during an ion pumping cycle. The amount of E1 and E2 Na/K-ATPase is regulated by extracellular K(+) and intracellular Na(+). Using purified enzyme preparations we find that the E1 Na/K-ATPase can bind both the Src SH2 and kinase domains simultaneously and keep Src in an inactive state. Conversely, the E1 to E2 transition releases the kinase domain and activates the associated Src. Moreover, we demonstrate that changes in E1/E2 Na/K-ATPase by either Na(+) or K(+) are capable of regulating Src and Src effectors in live cells. Together, the data suggest that the Src-coupled α1 Na/K-ATPase may act as a Na(+)/K(+) receptor, allowing salt to regulate cellular function through Src and Src effectors.     

Regulation of the frontocortical sodium pump by Na+ in Alzheimer's disease: difference from the age-matched control but similarity to the rat model

The Na+ and K+ dependence of the frontocortical Na,K-ATPase in Alzheimer's disease (AD) was compared with that in human control (Co) and rat AD model. In AD, the relationship between the Na/K ratio and the Na,K-ATPase activity showed noticeable left-shift with three-fold increase in the enzyme affinity for Na+ (K(0.5)=10 and 30 mM in AD and Co, respectively). The Na+ dependence of the enzyme in AD showed two different Hill coefficients (n(H)), 1.1 and 0.3, whereas the Co value of n(H) was higher (1.4). The rat AD model generated by ibotenic acid revealed a Na+ dependence similar to AD. The K+ dependence of the Na,K-ATPase showed no significant difference in AD and Co. Compared with Co, AD produced a shift in the break of the Na,K-ATPase Arrhenius plot, suggesting remarkable alterations in the enzyme lipid environment. Our findings support the hypothesis that dysfunction of the Na,K-ATPase in AD is provoked by altered Na+ dependence of the enzyme. An impairment of the pump functionality might serve as an early mechanism of AD that should be interrupted by selective pharmacological agents.     

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na⁺/K⁺ ion binding site of the Na,K-ATPase α subunit.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Epitope and mimotope for an antibody to the Na, K-ATPase.

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.     

Binding of Na+ ions to the Na,K-ATPase increases the reactivity of an essential residue in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI), a new imidazole-based probe, results in irreversible loss of enzymatic activity. Inactivation of 95% of the Na,K-ATPase activity is achieved by the covalent binding of 1 molecule of [3H]NIPI to a single site on the alpha-subunit of the Na,K-ATPase. The reactivity of this site toward NIPI is about 10-fold greater when the enzyme is in the E1Na or sodium-bound form than when it is in the E2K or potassium-bound form. K+ ions prevent the enhanced reactivity associated with Na+ binding. Labeling and inactivation of the enzyme is prevented by the simultaneous presence of ATP or ADP (but not by AMP). The apparent affinity with which ATP prevents the inactivation by NIPI at pH 8.5 is increased from 30 to 3 microM by the presence of Na+ ions. This suggests that the affinity with which native enzyme binds ATP (or ADP) at this pH is enhanced by Na+ binding to the enzyme. Modification of the single sodium-responsive residue on the alpha-subunit of the Na,K-ATPase results in loss of high affinity ATP binding, without affecting phosphorylation from Pi. Modification with NIPI probably alters the adenosine binding region without affecting the region close to the phosphorylated carboxyl residue aspartate 369. Tightly bound (or occluded) Rb+ ions are not displaced by ATP (4 mM) in the inactivated enzyme. Thus modification of a single residue simultaneously blocks ATP acting with either high or low affinity on the Na,K-ATPase. These observations suggest that there is a single residue on the alpha-subunit (probably a lysine) which drastically alters its reactivity as Na+ binds to the enzyme. This lysine residue is essential for catalytic activity and is prevented from reacting with NIPI when ATP binds to the enzyme. Thus, the essential lysine residue involved may be part of the ATP binding domain of the Na,K-ATPase.     

The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin.

A new rapid and sensitive method for characterizing lectin specificity using streptavidin-biotinylglycans as a tool is presented. This assay is analogous to enzyme immunoassay and takes advantage of the strong, irreversible adsorption of streptavidin to the wells of the chambers of titer plates. A series of streptavidin-biotinylglycans was first coated on a microtiter plate, and then one of six lectins, concanavalin A, wheat germ agglutinin, Phaseolus vulgaris (red kidney bean) erythro-agglutinin, Lens culinaris (lentil) agglutinin, Datura stramoniun agglutinin, or Sambucus nigra (elderberry bark) agglutinin coupled to horseradish peroxidase, was added. After incubation and thorough washing, only the lectin bound to a complementary glycan remained and could be detected and quantified by the peroxidase reaction. It was established that the lectins retained their oligosaccharide-binding specificities after coupling to the peroxidase, that the binding was inhibited by addition of the corresponding sugar inhibitors, and that the color intensity produced by the enzyme reaction is proportional to the amount of lectin-peroxidase bound to biotinylglycan complexed with streptavidin immobilized on the plate. As an example, it was found that the peroxidase-D. stramoniun agglutinin conjugate strongly bound biotinylglycans, GlcNAc3-Man5-R, GalGlcNAc3Man5-R, and GlcNAc3-4Man3-R (R = GlcNAc2-[6-(biotinamido)hexanoyl]-Asn). As little as 10 pmol/ml of lectin was detected. With the growing availability of biotinylglycans, the method should represent a reliable and simple procedure for screening lectin-oligosaccharide recognition qualitatively and quantitatively.     

Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.