Effect of sodium, lithium and amiloride on the K+-dependent phosphatase activity of membrane preparations of Na,K-ATPase

Effects of sodium, lithium and amiloride on the ATPase reaction and on its potassium-dependent step were studied using membrane preparations of Na,K-ATPase. It was established that the addition of 70 mM NaCl or LiCl to the reaction medium diminished the hydrolysis of para-nitrophenyl phosphate (pNPP) by 70 and 40%, respectively. Amiloride (0.8 mM) inhibited activities of Na,K-ATPase and pNPPase by 50 and 15%, respectively. The higher concentrations of amiloride produced a more prominent inhibition of Na,K-ATPase, but not of pNPPase. There was no correlation between the effect of amiloride on the pNPP hydrolysis and potassium concentration in the medium. There was the additivity in the inhibition of pNPPase by 0.8 mM amiloride and sodium or lithium ions up to the concentrations of ions as high as 30 mM. A conclusion is made that the inhibition of Na,K-ATPase by amiloride is mediated through the modification of the sensitivity of the enzyme to sodium.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

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.     

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.     

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

• 1.1. Membrane-bound (Na⁺ + K⁺)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P₃) of the electrolyte.
• 2.2. The effect of Li⁺ and Ba²⁺ on (Na⁺ + K⁺)-ATPase activity of the two membrane fractions (P₂ and P₃) was analysed with respect to K⁺ and Mg²⁺ ions, after the I₅₀ estimation.
• 3.3. The kinetics of the reactions with these cations were investigated showing that Li⁺ inhibits P₂ uncompetitively and for P₃ presented a mixed type inhibition.
• 4.4. Ba²⁺ behaved as an hyperbolic mixed type inhibitor for P₂ and a linear mixed type inhibitor for P₃ fraction.

     

Mechanisms of detergent effects on membrane-bound (Na+ + K+)-ATPase

Because the nonionic detergent octaethylene glycol dodecyl ether has been used extensively for studies on active solubilized preparations of (Na+ + K+)-ATPase, we tried to see if the detergent alters the properties of the membrane-bound enzyme prior to solubilization. Addition of the detergent, at concentrations below its critical micellar concentration, to reaction mixtures containing the highly purified membrane-bound enzyme reduced the K0.5 of ATP for (Na+ + K+)-dependent ATPase activity without affecting the maximal velocity or abolishing the negative cooperativity of the substrate-velocity curve. Under these conditions, however, the enzyme was not solubilized as evidenced by complete sedimentation of the membrane fragments containing the enzyme upon centrifugation at 100,000 X g for 30 min. Other nonsolubilizing effects of the detergent included an increase in K0.5 of K+, inhibition of Na+-dependent ATPase with no effect on K0.5 of ATP for this activity, and reductions in the spontaneous decomposition rates of the K+-sensitive phosphoenzyme obtained from ATP and the phosphoenzyme obtained from Pi. The nonsolubilizing effects of the detergent on the purified enzyme were obtained with no detectable lag, were readily reversible, and could be distinguished from its vesicle-opening effects on crude membrane preparations. Several other nonionic and ionic detergents had similar effects on the enzyme. The findings indicate (a) detergent binding to hydrophobic sites on extramembranous segments of enzyme subunits; (b) that occupation of these sites mimics the effects of ATP at a low-affinity regulatory site with no effect on high-affinity ATP binding to the catalytic site; and (c) that in studies on detergent-solubilized preparations, it is necessary to distinguish between the effects of solubilization per se and detergent effects at the regulatory site.     

Effect of glycosphingolipids on erythrocyte Na+,K+-ATPase activity

The total fractions of gangliosides and cerebrosides isolated from the tissue of human brain were studied for their effect on the Na+, K+-ATPase activity of native erythrocytes and their membranes. It is shown that gangliosides depending on time of their preincubation with the enzyme preparation and concentration produce both the activating and inhibiting action and cerebrosides--only the inhibiting one. Gangliosides inhibit the transport ATPase activity noncompetitively with respect to ATP and Na+ and competitively--to K+, cerebrosides inhibit it noncompetitively with respect to all ATPase activators.     

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.     

Effect of amiodarone on Na+-, K+-ATPase and Mg2+-ATPase activities in rat brain synaptosomes

Amiodarone hydrochloride is a diiodinated antiarrhythmic agent widely used in the treatment of cardiac disorders. With the increasing use of amiodarone, several untoward effects have been recognized and neuropathy following amiodarone therapy has recently been reported. The present studies were carried out to study the effect of amiodarone on rat brain synaptosomal ATPases in an effort to understand its mechanism of action. Na+, K+-ATPase and oligomycin sensitive Mg2+ ATPase activities were inhibited by amiodarone in a concentration dependent manner with IC50 values of 50 microM and 10 microM respectively. [3H]ouabain binding was also decreased in a concentration dependent manner with an IC50 value of 12 microM, and 50 microM amiodarone totally inhibited [3H]ouabain binding. Kinetics of [3H]ouabain binding studies revealed that amiodarone inhibition of [3H]ouabain binding is competitive. K+-activated p-nitrophenyl phosphatase activity showed a maximum inhibition of 32 per cent at 200 microM amiodarone. Synaptosomal ATPase activities did not show any change in rats treated with amiodarone (20 mg kg-1 day-1) for 6 weeks, when compared to controls. The treatment period may be short, since the reported neurological abnormalities in patients were observed during 3-5 years of treatment. The present results suggest that amiodarone induced neuropathy may be due to its interference with sodium dependent phosphorylation of Na+, K+-ATPase reaction, thereby affecting active ion transport phenomenon and oxidative phosphorylation resulting in low turnover of ATP in the nervous system.     

Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase

In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.     

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.     

Thermoinactivation and aggregation of alpha beta units in soluble and membrane-bound (Na,K)-ATPase

Stability and conformational transitions of soluble and fully active alpha beta units of (Na,K)-ATPase in n-dodecyl octaethylene glycol monoether (C12E8) are examined. Sedimentation equilibrium centrifugation gave a molecular weight of 143 000 for the alpha beta unit eluting from TSK 3000 SW gel chromatography columns. Fluorescence analysis and phosphorylation experiments show that E1-E2 transitions between both dephospho and phospho forms of soluble (Na,K)-ATPase are similar to those previously observed in the membrane-bound state. The two conformations can also be identified by their different susceptibilities to irreversible temperature-dependent inactivation. E1 forms of both soluble and membrane-bound (Na,K)-ATPase are more thermolabile than E2 forms. Gel chromatography on TSK 3000 SW and 4000 SW columns shows that thermal inactivation of soluble (Na,K)-ATPase at 40 degrees C is accompanied by aggregation of alpha beta units to (alpha beta)2 units and higher oligomers. The aggregates are stable in C12E8 but dissolve in sodium dodecyl sulfate. Similar aggregation accompanies inactivation of membrane-bound (Na,K)-ATPase at 55-60 degrees C. These data suggest that inactivation both in the soluble and in the membrane-bound state involves exposure of hydrophobic residues to solvent. The instability of the soluble E1 form may be related to inadequate length of the dodecyl alkyl chain of C12E8 for stabilization of hydrophobic protein domains that normally associate with alkyl chains of phospholipids in the membrane. Interaction between alpha beta units-does not seem to be required for the E1-E2 conformational change, but irreversible aggregation appears to be a consequence of denaturation of (Na,K)-ATPase in both soluble and membranous states.     

Molecular species of a soluble nucleoside diphosphokinase related to the Na + ,K + -ATPase

Soluble nucleosidediphosphokinase (NDK) activity is prepared from purified kidney membranes rich in Na⁺, K⁺-ATPase. The soluble NDK differs from the bound activity by the lack of sensitivity to Na⁺, K⁺ and ouabain. Sucrose gradient centrifugation of the soluble NDK shows the presence of three major species with molecular weights of 21,000, 92,000 and 138,000. Increasing the urea concentration in the sucrose gradient centrifugation decreases the proportion of the enzyme in the heavier form. This effect of urea is reversible. These data suggest that the higher molecular weight forms are tetramers and hexamers of the 21,000 dalton form of the soluble NDK.     

Comparison of detergent-solubilized and membrane-bound forms of kidney (Na + K)-ATPase

The detergent solubilization of dog kidney (Na + K)-ATPase has been investigated. The nonionic detergents, Brij 58, C₁₂E₈, and Lubrol WX were tested for their ability to produce active, soluble enzyme. Lubrol WX gave the best results. Enzyme so treated is found in the supernatant fraction after centrifugation at 100,000g for 1 h. It has the same or slightly greater specific activity, the same subunit composition as judged by SDS-gel electrophoresis, and very similar kinetic parameters with respect to sodium, potassium, ATP, pNPP, and ouabain as the membrane-bound enzyme. The Lubrol-treated enzyme is stable for at least 5 days at 4 °C. The phospholipid content of the Lubrol-treated enzyme is decreased, as might be expected, by about 50%. Limited tryptic proteolysis and fluorescence changes seen after modification with FITC indicate that the solubilized (Na + K)-ATPase undergoes the same conformational transitions as the membrane enzyme. Our results indicate that kidney enzyme solubilized as described here is nondenatured and fully active, and therefore a valuable preparation for spectroscopic and other approaches for study of this enzyme.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

久效磷对美国红鱼鳃Na^＋／K^＋-ATP酶活性和超显微结构的影响

经不同试验浓度的久效磷(0．25、0．5、1．0和2．0mg·L^-1)处理美国红鱼4d后,分别对鱼鳃Na^+／K^+—ATP酶活性和氯细胞密度进行了测定和计数,并观察了鱼鳃组织显微结构和超微结构的变化。结果表明,低浓度久效磷(0．25mg·L^-1)处理可以诱导鱼鳃氯细胞大量增生,Na^+／K^+—ATP酶活性增强,随着试验浓度的增加,久效磷对鳃组织的损伤越来越重,Na^+／K^+—ATP酶活性逐渐降低;久效磷对鱼鳃显微结构的损伤表现为鳃小片上皮细胞水肿、脱离。鳃小片基部粘连。鳃小片上皮细胞角质化;超微结构变化主要为内质网、线粒体、微小管和核膜的水肿及部分溶解,这种损伤表现为由细胞膜到细胞核的动态过程。     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes

The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.