Variations in (Na+ + K+)-ATPase and Mg2+-ATPase activity of the ground squirrel brain during hibernation.

1. The specific activity of brain (Na+ + K+)-ATPase and Mg2+ -ATPase of the ground squirrel (Spermophilus richardsonii) is significantly increased after long-term hibernation. 2. The markedly non-linear thermal dependence of (Na+ + K+)-ATPase is unchanged during hibernation whereas the near linear thermal dependence of Mg2+-ATPase undergoes minor alteration after prolonged hibernation. 3. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is significantly decreased after 100 days of hibernation as is both the rate and amount of [3H]-ouabain binding. 4. These changes may be related to alteration in the phospholipid matrix of the membrane rather than alteration in the protein structure of the enzyme.     

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.     

Mg2+-induced changes of lipid order and conformation of (Na+ + K+)-ATPase

The effect of magnesium on the phospholipid order parameter and not the conformation of purified pig kidney outer medulla (Na+ + K+)-ATPase was investigated by fluorescence techniques. Measurements with a fluorescent probe TMA-DPH and its sensitized fluorescence with tryptophan residues as donors revealed that magnesium increased the order of the membrane phospholipids both in the lipid annulus and in the bulk phase. Changes in the lipid order induced by Mg2+ can be closely referred to the protein arrangement followed by the steady-state anisotropy of FITC-labeled (Na+ + K+)-ATPase.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Inhibitory action of phosphatidylinositol on synaptosomal (Na+ + K+)-ATPase activity

Phosphatidylinositol and several other phospholipids were tested for their ability to influence the (Na+ + K+)-ATPase activity of the cortical synaptic membrane from rats at various levels of free Ca2+. Phosphatidylinositol, but not phosphatidylethanolamine, phosphatidylcholine nor phosphatidylserine, markedly inhibited this enzyme activity, when the free Ca2+ concentration in the incubation media was less than 2.5 X 10(-6) M. This result suggests that phosphatidylinositol may play a role in the depolarization and/or the release of neurotransmitters or intracellular substances in the brain.     

Expression of functional (Na+ + K+)-ATPase from cloned cDNAs

Functional (Na+ + K+)-ATPase is formed in Xenopus oocytes injected with alpha- and beta-subunit-specific mRNAs derived from cloned Torpedo californica cDNAs. Both the mRNAs are required for the expression of functional (Na+ + K+)-ATPase.     

Crystallization patterns of membrane-bound (Na+ + K+)-ATPase

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric αβ-unit of the enzyme protein. In phosphate-induced crystals an $\text{(αβ)}{}_2\text{-}\text{unit}$ occupies one unit cell suggesting that interactions between αβ-units can be of importance in the function of the Na⁺, K⁺ pump.     

Crystallization patterns of membrane-bound (Na+ +K+)-ATPase

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.     

Potassium binding to the (Na+ + K+)-ATPase

The number of K+ bound to the (Na+ + K+)-ATPase has been measured under equilibrium conditions by a differential-titration technique (Hastings, D.F. (1977) Anal. Biochem. 83, 416-432). 5.1 K+ were bound per 32P-labelling site. The K'D for K+ was dependent on the concentration of choline, which was included to give ionic strength. K'D was 59 +/- 2.5 microM with 97 mM choline, 26 +/-1.9 microM with 30 mM choline. The K+ : choline selectivity was 2564 : 1 and the calculated K'D for K+ with zero choline was 11 microM and for choline with zero K+ was 28 mM. 20 microM ATP in the presence of 97 mM choline incresed the K'D for potassium 3-fold to 177 +/- 14 microM. The K'D for K+ with 3 mM Na+ in the presence of 27 mM choline was 81 +/- 10 microM and with 30 mM Na+ without choline 700 +/- 250 microM. The calculated K'D for Na+ at zero K+ and zero choline was 0.6 +/- 0.2 mM. The K+ : Na+ selectivity was 54 : 1.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations.