Activation of rabbit brain microsomal (Na+ plus K+)-dependent ATPase by a leukocytic product.

The properties of a (Na+ plus K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activator contained in leukocytic extracts was investigated. Intact polymorphonuclear leukocytes release the activator in a time- and temperature-dependent process. It is non-dialyzable through cellophane; inactivated by protease, trypsin, or phenol; contains essential sulfhydryl groups; and is heat and acid labile. Treatment of ATPase with the activator and subsequent removable of the activator from mixtures did not reverse the ATPase activation.     

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.     

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.     

Mechanisms by which Li+ stimulates the (Na+ and K+)-dependent ATPase.

The addition of LiCl stimulated the (Na+ + K+)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na+/low K+ media and at low Mg-ATP concentrations. Apparent affinities for Li+ were estimated at the alpha-sites (moderate-affinity sites for K+ demonstrable in terms of activation of the associated K+-dependent phosphatase reaction), at the beta-sites (high-affinity sites for K+ demonstrable in terms of activation of the overall ATPase reaction), and at the Na+ sites for activation. The relative efficacy of Li+ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li+ was substituted for K+, and also in terms of the relative effect of Li+ on the apparent Km for Mg-ATP. With these data, and previously determined values for the apparent affinities of K+ and Na+ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li+ stimulates by relieving inhibition due to K+ and Na+ (i) by competing with K+ for the alpha-sites on the enzyme through which K+ decreases the apparent affinity for Mg-ATP and (ii) by competing with Na+ at low-affinity inhibitory sites, which may represent the external sites at which Na+ is discharged by the membrane Na+/K pump that this enzyme represents. Both these sites of action for Li+ would thus lie, in vivo, on the cell exterior.     

The pre-steady-state hydrolysis of ATP by porcine brain (Na+ + K+)-dependent ATPase.

The hydrolysis of [gamma-32P]ATP by porcine brain (Na+ + K+)-stimulated ATP phosphohydrolase (EC 3.6.1.3) has been studied at 28 degree C in a rapid mixing quenched-flow apparatus. An "early burst" in the release of Pi from ATP has been observed when the enzyme is mixed with ATP, Na+ and a relatively high concentration of K+ (10 mM) but the burst is less pronounced with 0.5 mM K+. This "early burst" of Pi release is suppressed when the enzyme is pre-mixed with 10 mM K+ or 20% (v/v) dimethylsulphoxide before mixing with ATP and Na+, and premixing of enzyme with Na+ antagonizes this effect of dimethylsulphoxide. The results have been analysed by a non-linear least squares regression treatment and are consistent with a mechanism involving three steps, one of which may be a relatively slow change in enzyme conformation following release of Pi from its covalent linkage with the enzyme, in addition to formation of the enzyme-substrate complex. Rate constants (and S.E.) for these steps have been calculated and the roles of phospho-enzyme and other intermediates in the reaction mechanism of the transport ATPase are dicussed.     

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.     

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+.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

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.     

The effect of chelators on Mg2+, Na+-dependent phosphorylation of (Na+ + K+)-activated ATPase.

1. The effect of free Mg2+, MgEDTA and MgCDTA on the phofphorylation of the (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been studied. 2. 10 mM trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) added simultaneously with [gamma-32P]ATP to a solution containing the enzyme, 1 mM Mg2+ and 150 mM Na+ does not prevent formation of phospho-enzyme. When [gamma-32P]ATP is added after CDTA the level of phospho-enzyme obtained decreases with increase in the time interval between addition of CDTA and ATP. The inability of CDTA to prevent the formation of phospho-enzyme becomes more pronounced when the medium contains MgEDTA. In the presence of CDTA the maximum amount of phospho-enzyme formed increases with the MgEDTA concentration. 3. Without CDTA the steady-state level of phospho-enzyme is directly proportional to the logarithm of free Mg2+ concentration. Neither with suboptimal nor with optimal concentrations of free Mg2+ does MgEDTA have an effect on the level of phospho-enzyme formed. 4. Using the phospho-enzyme level as a measure of free Mg2+ the experiments show that CDTA reacts slower with Mg2+ than does EDTA, but the stability constant of MgCDTA complex is higher than of MgCDTA, complex. 5. Due to the higher stability constant, of MgCDTA, as compared to MgEDTA, addition of CDTA to a medium containing free Mg2+ and MgEDTA will not only chelate the free Mg2+, but it will also shift the equilibrium from MgEDTA towards MgCDTA, i.e. MgEDTA acts as a source of free Mg2+ which is then chelated by CDTA. The experiments show that it takes minutes before Mg2+, EDTA and CDTA come to equilibrium. Provided the dissociation of MgEDTA is faster than the formation of the MgCDTA complex, the medium will contain a concentration of free Mg2+ which at any given instant is near in equilibrium with a slowly decreasing concentration of MgEDTA; this free Mg2+ can support phosphorylation. This can explain why the rate with which CDTA stops phosphorylation decreases with an increase in the MgEDTA concentration. 6. When phosphorylation is stopped by addition of unlabelled ATP, the rate of dephosphorylation is faster than when it is stopped by addition of CDTA both with and without EDTA in the medium. CDTA reacts too slowly with Mg2+ to be used as a chelator in studies where a fast removal of Mg2+ is required. 7. A previous finding has been verified, namely that the rate of spontaneous, of K+-stimulated and of ADP-stimulated dephosphorylation is independent of the Mg2+ concentration during formation of phospho-enzyme.     

A model for the reaction pathways of the K+-dependent phosphatase activity of the (Na+ + K+)-dependent ATPase

(Na+ + K+)-dependent ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+ -dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: (formula; see text) Differences between the three preparations in K0.5 for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in K0.5 produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+ -dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+ -sites are oriented extracellularly, as are those of the (Na+ + K+)-dependent ATPase reaction. Dimethyl sulfoxide accentuated Na+ -stimulated K+ -dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+ -sites of the Na+ -stimulated pathway.     

Transport ATPase: thimerosal inhibits the Na+ +K+-dependent ATPase activity without diminishing the Na+-dependent ATPase activity.

A guinea pig kidney membrane preparation was incubated with thimerosal and then thoroughly washed. Comparison of the properties of the native and the modified membranes showed that (a) Na⁺+K⁺-dependent activity is substantially inhibited by thimerosal; (b) thimerosal does not diminish Na⁺-dependent ATPase activity; and (c) the thimerosal treated enzyme, like the native enzyme, is phosphorylated in the presence of Na⁺ and ATP, and dephosphorylated upon the addition of K⁺. It is suggested that thimerosal does not affect the binding of ATP to the high-affinity catalytic site, but that it blocks the binding of ATP to a low affinity modifying site the occupation of which is essential for the dissociation of the stable K⁺-dephosphoenzyme and the recycling of the enzyme.