Identification of a disulfide between cysteine 214 and cysteine 277 in the beta subunit of native (Na+ + K+)ATPase

Two peptides, produced during tryptic digestion and thermolytic digestion, respectively, and containing the same intact disulfide from the beta polypeptide of (Na+ + K+)ATPase from Torpedo californica, were isolated and unambiguously identified. The disulfide is between Cysteine 214 and Cysteine 277.     

Inhibition of erythrocyte membrane (Na+ + K+)-activated ATPase by ozone-treated phospholipids.

Ozone-treated aqueous suspensions of natural phospholipids yield at least two types of inhibitors of human erythrocyte membrane (Na+ + K+)-ATPase. The more labile ones appear to be carbonyl-containing substances whose inhibitory properties are enhanced if ozonolysis takes place in the presence of putrescine or glycine. Other amines of similar structure are much less effective as potentiators. Semicarbazide destroys the inhibitory properties of the more labile substances and can release putrescine from the complexes it forms with the carbonyl products of ozonolysis. 3the more stable inhibitors are unaffected by putrescine, glycine, or semicarbazide. Synthetic, saturated phospholipids do not produce these inhibitors during ozonolysis.     

Substrate sites for the (Na+ + K+)-dependent ATPase.

Kinetic studies on a rat brain (Na+ + K+)-dependent ATPase (EC 3.6.1.3) preparation demonstrated high-affinity sites for ATP, with a Km near 1 mum, and low affinity sites for ATP, with a Km near 0.5 mM. In addition, the dissociation constant for ATP at the low affinity sites was approached through the ability of ATP to modify the rate of photo-oxidation of the enzyme in the presence of methylene blue; a value of 0.4 mM was obtained. The temperature dependence of the Km values in these two concentration ranges also differed markedly, and the estimated entropy of binding was +27 cal/degree per mol at the high affinity sites, whereas it was -20 cal/degree per mol at the low affinity sites. Moreover, the relative affinities of various congeners of ATP as of the K+ -dependent phosphatase reaction of the enzyme indicated an interaction at the low-affinity sites for ATP: ATP, ADP, CTP, and the [beta-gamma] -imido analog of ATP all competed with Ki values near those for the ATPase reaction at the low affinity sites. Conversely, the Km for nitrophenyl phosphate as a substrate for the phosphatase reaction was near its Ki as a competitor at the low-affinity sites of the ATPase reaction. These observations are incorporated into a reaction scheme with two classes of substrate sites on a dimeric enzyme, manifesting idverse enzymatic and transport characteristics.     

Interaction of palytoxin and cardiac glycosides on erythrocyte membrane and (Na+ + K+) ATPase

Palytoxin (PTX), at extremely low concentrations (0.01-1 nM), caused K+ release from rabbit erythrocytes. Among the various chemical compounds tested, cardiac glycosides potently inhibited the PTX-induced K+ release. The order of inhibitory potency (IC50) was cymarin (0.42 microM) greater than convallatoxin (0.9 microM) greater than ouabain (2.3 microM) greater than digitoxin (88 microM) greater than digoxin (90 microM). Their corresponding aglycones, even at 10 microM, did not inhibit the K+ release, but competitively antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex in narrow concentration ranges (IC50 = 0.15-2.4 microM), suggesting that the inhibition of K+ release is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity, and the sugar moiety of cardiac glycosides is involved in the inhibition. On the other hand PTX, at higher concentrations (greater than 0.1 microM), inhibited the (Na+ + K+)-ATPase activity. However, this inhibitory effect of PTX was not antagonized by ouabain. It is suggested that, compared with ouabain, PTX has additional binding site(s) on the (Na+ + K+)-ATPase.     

Ouabain-resistant human lymphoblastoid lines altered in the (Na+ + K+)-dependent ATPase membrane transport system.

Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)-dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 muM ouabain. Ouabain-resistant (OUA(R)) cells with normal growth at 50 times this dose were recovered at a frequency 1 X 10(-6). This frequency was increased 9-fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR-191, under conditions where both increased the frequency of 8-azaguanine-resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUA(R) clones show showed 30-50% of the wild type amount of 3H-ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 muM at 0.5 mM K+, as observed in wild-type cells. Both the initial rate of uptake of 86Rb+ in OUA(R) cells and the (Na+ + K+)-dependent ATPase activity of OUA(R) plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUA(R) cells in the absence of ouabain were unchanged compared with wild type cells.     

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.     

Assembly of the (Na+ + K+)-adenosine triphosphatase. Post-translational membrane integration of the alpha subunit

Guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates and wheat germ extracts. Antibodies to the holoenzyme (Na/K-ATPase) immunoprecipitated only a 96,000-dalton product which was identified as the alpha subunit with a molecular weight that was indistinguishable from that of mature alpha subunit. To explore the possibility that the primary translational product is integrated as such into membranes, guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates in the presence of dog pancreas microsomes; two immunoprecipitated products were detected, the 96,000-dalton alpha subunit and a 135,000-dalton new component that was integrated into the microsomal membrane since it was completely resistant to extraction with alkali. Addition of purified alpha subunit inhibited the binding of antibody to the 135,000-dalton product and extraction with urea-sodium dodecyl sulfate recovered the 96,000-dalton product, implying that the 135,000-dalton product was an alpha-chi dimer. Translation of size-fractionated poly(A+) RNA yielded evidence that the 135,000-dalton product is encoded in two separate mRNAs. The integration in vitro of the alpha subunit is, therefore, dependent on the co-translational integration into the membranes of a smaller peptide (35,000 to 40,000 daltons) which is presumably the beta subunit. Evidence was also obtained that this mechanism is present in vivo by isolation of mRNA alpha from free polysomes, as well as detection of the cytosolic form of the alpha subunit in pulse-chase experiments in MDCK cells.     

Functionally distinct classes of K+ sites on the (Na+ + K+)-dependent ATPase.

K+ interactions with a rat brain (Na+ + K+)-dependent ATPase and the associated K+-dependent nitrophenyl phosphatase activity were examined. Classes of sites for K+ were distinguished, initially, on the basis of affinity estimated by kinetic analysis in terms of KO.5 (the concentration for half-maximal activation), and by K+-accelerated enzyme inactivation by F-minus, which permits evaluation of a dissociation constant for K+, KD. Moderate-affinity sites ("alpha sites"), with a KD near 1 mM, were demonstrable for the phosphatase activity and for the "free" enzyme. High-affinity sites ("beta sites"), with a KD near 0.1 mM, were seen for the overall ATPase activity and under conditions in which enzyme phosphorylation by substrate also occurs. Further differentiation between alpha and beta sites was made in terms of (i) the characteristic changes in affinity with pH, and (ii) the efficacy of Li+ relative to K+, Rb+, Cs+, and Tl+ at these two classes of sites. Low-affinity sites ("gamma sites") through which K+ inhibits enzymatic activity were also detectable, with a KD around 140 mM. These data are incorporated into a model for the reaction sequence to accommodate both transport processes and certain K+/ATP antagonisms.     

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.     

Interactions between K+ and ATP binding to the (Na+ + K+)-dependent ATPase.

K+ appears to decrease the affinity of the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, Mg2+ - ATP, and Mg2+ - ATP, in turn, appears to decrease the affinity of the enzyme for K+. These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K+ sites on the enzyme involved. K+ increased the apparent Km for Mg2+ - ATP, an effect that was antagonized competitively by Na+. The data can be fitted to a model in which Mg2+ - ATP binding is prevented by occupancy of alpha-sites on the enzyme by K+ (i.e. sites of moderate affinity for K+ accessible on the "free" non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these alpha-sites by Na+ has no effect on Mg2+ - ATP binding to the enzyme. On the other hand, Mg2+ - ATP decreased the apparent affinity of the enzyme for K+ at the alpha-sites, in terms of (i) the KD for K+ measured by K+-accelerated inactivation of the enzyme by F-, and (ii) the concentration of K+ for half-maximal activation of the K+-dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K+ from discharge sites, it is consistent with observations that K+ can inhibit the enzyme at low substrate concentrations, and that Li+, which has poor efficacy when occupying these alpha-sites, can stimulate enzymatic activity at high K+ concentrations by displacing the inhibitory K+.     

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.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Myometrial (Na+ + K+)-activated ATPase and its Ca2+ sensitivity

Ouabain-sensitive (Na+ + K+)-ATPase activity in the rat myometrial microsome fraction could only be determined following detergent treatment. The (Na+ + K+)-ATPase activity manifested by detergent treatment proved very stable even to high concentrations of NaN3, in contrast Mg+-ATPase activity was reduced to about 30 percent of the control. The major part of the Mg2+-ATPase in the myometrial membrane preparation was found to be identical with the NaN3-sensitive ATP diphosphohydrolase capable of ATP and ADP hydrolysis. This monovalent-cation-insensitive ATP hydrolysis could be extensively reduced by DMSO. Furthermore DMSO prevented the inactivation of the (Na+ + K+)-ATPase activity. 10-100 microM Ca2+ inhibited the (Na+ + K+)-ATPase activity obtained in the presence of SDS by 15-50 percent. The Ca2+ sensitivity of the enzyme was considerably decreased if the proteins solubilized by the detergent had been separated from the membrane fragments by ultracentrifugation. The inhibitory effect could be regained by combining the supernatant with the pellet. Ca2+ sensitivity of the (Na+ + K+)-ATPase activity was preserved even after removal of the solubilized proteins provided that DMSO had been applied. It appears that a factor in the plasma membrane solubilized by SDS may be responsible for the loss of Ca2+ sensitivity of the (Na+ + K+)-ATPase activity, the solubilization of which can be prevented by DMSO.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.