Hydrophobic labeling of (Na+,K+)-ATPase: further evidence that the beta subunit is embedded in the membrane bilayer

O-Hexanoyl-3,5-diiodo-N-(4-azido-2-nitro-phenyl)tyramine has been used after photochemical conversion into the reactive nitrene to label (Na+,K+)-ATPase from Bufo marinus toad kidney. Immunochemical evidence indicates that the reagent labels both subunits of the enzyme in partially purified form as well as in microsomal membranes. These results support the view that the glycoprotein subunit, like the catalytic subunit, possesses hydrophobic domains by which it is integrated into the plasma membrane.     

Na+-ATPase is a different entity from the (Na+ + K+)-ATPase in rat kidney basolateral plasma membranes

In this work, we present evidence in agreement with the hypothesis that there exist two Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells: (1) (Na+ + K+)-ATPase activity, which is inhibited by ouabain and by treating the membranes with trypsin, is insensitive to furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 1.6; (2) the Na+-ATPase activity, which is insensitive to ouabain and to trypsin treatment, is inhibited by furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 0.4.     

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.     

Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]

In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.     

Subcellular localization of (Na+ + K+)-activated ATPase in the brush border membrane of the mucosal cell of the rat small intestine.

The localization of (Na+ + K+)-activated ATPase was investigated in isolated brush borders of rat small intestinal mucosa. The purity of the fractions has been checked by morphological and enzymatic criteria. The brush borders were found to contain a significant quantity of (Na+ + K+)-activated ATPase. Separation of isolated brush borders into their substructures suggests that (Na+ + K+)-activated ATPase is localized deeper within the brush border region than invertase. These findings are discussed in relation to active monosaccharide transport in the intestine.     

Orientation of the beta subunit polypeptide of (Na+ + K+)ATPase in the cell membrane

Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.     

Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.     

Effects of polyamines on partial reactions of membrane (Na+ + K+)-ATPase.

Spermine and spermidine inhibit the (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) reaction so that the effect increases as the ionic content due to Na+ and K+ in the reaction is reduced. Several other amines inhibit (Na+ + K+)-ATPase to varying degress and methylglyoxal-bis-(guanylhydrazone) was the most potent inhibitor among those tested. The inhibition by polyamines of the ATPase is uncompetitive with respect to Mg2+ and ATP activation of the reaction. Various naturally occurring polyamines and other amines inhibited Na+ activation of (Na+ + K+)-ATPase as well as Na+-dependent phosphoenzyme formation in an apparently competitive manner with respect to Na+. Likewise, K+-activation of (Na+ + K+)-ATPase as well as K+-p-nitrophenyl phosphatase was inhibited in an apparently competitive manner with respect to K+. Both the cation charge and structure (e.g., aliphatic chain length) may contribute to the inhibitory effects of the amines; however, Na+ sites appear to be more sensitive to cation charge than the aliphatic chain length of the amine, whereas the opposite appears to be true for K+ sites. The results do not indicate a specific effect of polyamines on (Na+ + K+)-ATPase or its partial reactions.     

Effects of aldosterone on (Na+ + K+)-ATPase of amphibian sodium-transporting epithelial cells (A6) in culture

Stimulation by aldosterone of sodium reabsorption can be reproduced on a cell line, A6, derived from the renal tissue of Xenopus laevis. These cells organize themselves as a polarized epithelium carrying out unidirectional sodium transport, reflected by the short-circuit current (Isc). Isc response to aldosterone starts to be apparent after a latency period of 2-3 h; the full hormonal effect takes much longer. On the other hand, (Na+ + K+)-ATPase activity and density in ouabain binding sites did not increase before several hours of treatment. At that stage, while Isc more than trebled, Na+ pump activity and density went up by less than 50%. A significant influence of aldosterone on the way the Na+ pump operates is considered unlikely, since cell interaction with ouabain remained unchanged (Kd approximately 18 nM). Furthermore, the close correspondence of hormonal effect, in relative terms, on (Na+ + K+)-ATPase activity vs density, argues against a significant degree of recruitment of spare pump units. Thus aldosterone effect on Na+ pump probably results from increased biosynthesis of the enzyme. The aldosterone dependent Na+ pump stimulation is apparently unrelated to sodium available for transport. The hormone seems to act on Na+ pump directly.     

Immunochemical studies on the large polypeptide of electrophorus electroplax (Na+ + K+)-ATPase.

Antibodies against Lubrol-solubilized Electrophorus electroplax (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and its 96 000-dalton polypeptide (P96) were raised in rabbits. The P96 antibody does not cross react with the (Na+ + K+)-ATPase from mammalian species and tissues, but it cross reacts with the (Na+ + K+)-ATPase from both Electrophorus electroplax and brain. The combination of enzyme with anti-P96 is found to inhibit phosphoryl enzyme formation to the same extent that it inhibits enzyme activity. The rate of K+-sensitive dephosphorylation of phosphoryl enzyme appears to be unchanged. These are also found to be true with the antibody against the whole enzyme. Upon tryptic digestion of the enzyme-anti-P96 complex only the large polypeptide of the enzyme is protected. In the case of enzyme-anti-Lubrol-solubilized enzyme complex, both the large and small polypeptides are protected, whereas preimmune sera are without any protecting effect. The data indicate that the phosphoryl acceptor polypeptide and the Lubrol-solubilized electroplax (Na+ + K+)-ATPase from which the polypeptide is derived are phylogenetically distinct from those of the mammalian (Na+ + K+)-ATPases. The selective tryptic resistance of the enzyme-anti-P96 complex indicates that the two polypeptides are spatially well separated, possibly on opposite sides of the membrane.     

Influence of certain fish meals on (Na+ + K+)-ATPase and Ca2+-ATPase activity in rat small intestine

The effect of high-protein content fish meal on (Na+ + K+)-ATPase and Ca2+-ATPase activity in rat small intestine was studied. 5 groups of Wistar rats, weighing between 40-60 g, were fed diets with 12% protein content of dry matter for 10 days. The protein source was casein for the control group and fish meal derived from Coryphaenoides rupestris, Chimaera monstruosa and Merluccius merluccius for the test group. The results show a decrease in (Na+ + K+)-ATPase and a rise in Ca2+-ATPase activity in animals fed with fish meal protein compared to those fed on casein. No significant variations were observed between the groups fed on fish meal derived from C. rupestris and Ch. monstruosa. The calcium ion, which is abundant in fish, may be a factor responsible for these variations which produce inhibition of the (Na+ + K+)-ATPase and stimulation of the Ca2+-ATPase.