Effects of sodium and potassium ions on the elementary steps in the reaction of Na+-K+-dependent ATPase1.

The effects of Na+ and K+ ions on the elementary steps in the reaction of Na+-K+-dependent ATPase (EC 3.6.1.3) were investigated in 0.5-600mM NaCL and 0-10mM KCL, at a fixed concentration (1mM) OF MgCL2, AT PH 8.5 and at 15 degrees. The data were analyzed on the basis of the reaction mechanism in which a phosphorylated intermediate, E ADP P (abbreviated as EP), is formed via two kinds of enzyme-substrate comples, E1ATP and E2ATP, and EP is in equilibrium with E2ATP, and is hydrolyzed to produce P1 and ADP. The following results were obtained: 1. The rate od E2ATP-formation, vf, increased with increase in the Na+ concentration, reached a maximum level, and then decreased with further increase in the Na+ concentration at various K+ concentrations. The value of vf was given as (see article). 2. The reciprocal of the equilibrium constants, K2, of the step E1ATPEQUILIBRIUM E ADP P in the presence of low concentrations of Na+ was larger than that in the presence of high concrntrations of Na+, indicating that the equilibrium shifted markedly toward E2ATP at low concentrations of Na+. The relation of K3 with Na concentration was rather complicated on varying the concentration of K+. However, generally speaking, it increased with increase in the K+ concentration. 3. The decomposition of EP was markedly activated by even low concentrations of K+, and inhibited by high concentrations of Na+. The inhibition by Na+ was partially suppressed by K+. The rate constant of EP-decomposition, vo/(EP), was given by (see article) where (vo/(EP) K+EQUALS0 was the value of vo/[EP] in the absence of K+.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

Properties of the conversion of an enzyme-ATP complex to a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase1.

Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article).     

Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells

Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chem., 243:1957), the phosphoenzyme formed at 0 degrees C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested that the conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1 approximately P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0 degrees C. Since the ATP:ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1 approximately P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0 degrees C. Simple saturation was observed instead with a K0.5 for Na of approximately 1 mM. The effect of increasing temperature on the ATP:ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0 degrees C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0 degrees C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 degrees C, there was an approximately 300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the E1 approximately P----E2P transition of the Na pump in human red cell membranes is blocked at 0 degrees C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP:ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0 degrees C, where E1 approximately P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme.     

Order of release of ADP and Pi from phosphoenzyme with bound ADP of Ca2+-dependent ATPase from sarcoplasmic reticulum and of Na+, K+-dependent ATPase studied by ADP-inhibition patterns

The inhibition of Ca2+-dependent ATPase from SR [EC 3.6.1.3] by ADP was of mixed type under both low Ca2+ and high Mg2+ concentration and high Ca2+ and low Mg2+ concentrations. On the other hand, the inhibition of Na+, K+-dependent ATPase [EC 3.6.1.3] by ADP was of competitive type in the presence of low and high K+ concentrations. These results suggest that ADP is released before Pi from the phosphoenzyme with bound ADP (EPADP) in the case of Ca2+-ATPase, but that Pi is released before ADP in the case of Na+, K+-ATPase.     

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.     

Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. III. Marked changes in affinities for monovalent cations induced by formation of an ADP-insensitive but no an ADP-Sensitive phosphoenzyme

We measured the amounts of Rb+ ions (a K+ congener) as well as Na+ and K+ ions bound to the ATPase during the ATPase reaction at pH 7.5 and 0 degrees C. The affinity of the Na+-binding sites for three Na+ ions decreased markedly but that of the K+-binding sites for two K+ or Rb+ ions increased markedly upon formation of an ADP-insensitive phosphorylated intermediate. Furthermore, the present experiment did not give any indication of a change in the Hill coefficient of 2, and showed an increase in the affinity of the K+-binding sites for Rb+ ions of about 28 times upon the formation of an ADP-insensitive EP. The enzyme state with a high affinity for Rb+ was maintained after the disappearance of EP. When the ATPase was treated with N-ethylmaleimide (NEM), almost all the EP formed was ADP-sensitive. The formation of an ADP-sensitive EP with the NEM-treated enzyme induced no change in the affinities of the ATPase for Na+ and Rb+ ions.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Insulin regulates ionic metabolism in a fresh water teleost, anabas testudineus (bloch)

Short term effects of insulin on total brain and branchial Na+K+ ATPase, Ca2+ ATPase and Na+, K+ and Ca2+ ions were investigated in A. testudineus. The increase in brain Ca2+ ATPase after alloxan treatment may account for an increased amount of intracellular calcium required for biochemical events taking place inside the cells. Branchial Na+K+ATPase was significantly stimulated while Ca2+ ATPase significantly inhibited after alloxan treatment. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca2+ transport via; its action on the Ca2+ pump protein rather than the membrane permeability to Ca2+. The increased activity of brain Na+K+ ATPase at 3 and 24 hr by insulin to alloxan pretreated fish may account for the stimulated co-transport of glucose and its utilization for energy requirements and the excitatory action on neurons in the brain. The elevated brain Ca2+ ATPase may be due to the role of calcium as a second messenger in hormone action. At 24 hr, the activity of branchial Na+K+ ATPase and Ca2+ ATPase in alloxan pretreated specimens was significantly stimulated by insulin. This may be due to increased synthesis of these enzyme units. Administration of insulin (lU/fish) in normal fish significantly inhibited the activity of brain and branchial Na+K+ ATPase while brain Ca2+ ATPase showed a stimulatory effect at 3 and 24 hr compared to control. Inhibition of total branchial Ca2+ ATPase activity by insulin may be due to increased Ca2+ concentration. Higher plasma glucose level in alloxan treated groups confirms the diabetic effect of alloxan. Insulin reverses this effect. The possible mechanism by which insulin controls Na+K+ ATPase activity appears to be tissue specific. The results seem to be the first report on the effect of insulin on ATPase activity in a teleost. These data are consistent with the hypothesis that insulin performs a role in hydro mineral regulation in freshwater teleosts.     

HPLC method for measuring (Na(+)-K(+)) ATPase and (Ca(++)-Mg(++)) ATPase in erythrocytes from different species of mammals]

(Na(+)-K+)ATPase and (Ca(++)-Mg++)ATPase are enzymes located in erythrocyte plasma membranes, driving back ions against the electrochemical gradient; (Na(+)-K+)ATPase transports 3 Na+ ions out of the cell, and 2 K+ ions into it for each hydrolyzed ATP molecule, whereas the Ca(2+)-pump transports Ca2+ ions out of the cells, by utilizing still the ATP hydrolysis. The method used to test the activity of the above mentioned enzymes is based on the measuring of the ADP quantity released during the reaction by HPLC, that is High Performance Liquid Chromatography; the chromatographic type is a Ion-Pair Reversed-Phase. This method presents the following important advantages for the assay of the enzymes we analysed: 1) It is reproducible through time; 2) It is perfectly linear; 3) It is extremely sensitive. This method allowed us to carry out a comparative study of (Na(+)-K+)ATPase and (Ca(++)-Mg++)ATPase in erythrocyte plasma membranes of several species of mammalia: man, horse, rabbit, lamb, rat. We recovered different values in ATPase activity; (Ca(++)-Mg++)ATPase shows a higher activity than Na(+)-K+)ATPase; moreover, some differences exist in the various Mammalia considered, with relation to each pump: the lamb shows the lowest activity for both pumps, whereas the rabbit shows the highest one. At present, the different values obtained are being interpreted and analysed. This method is also very versatile, since it allowed us to assess the Km value for Ca++ of the (Ca(++)-Mg++)ATPase in erythrocyte plasma membranes of rabbit. The value resulted to be 100 microMs, thus 10 times higher than the human Km value for the Ca++.     

The pre-steady state of Na+-K+-dependent ATPase after addition of Na+ ions. Transition of the phosphorylated intermediate from an ADP-sensitive to an ADP-insensitive form.

Na+-K+-Dependent ATPase [EC 3.6.1.3] was preincubated with ATP in the presence of a high concentration of MgCl2, and the phosphorylated intermediate, EP, was formed by adding a high concentration of NaCl. The following results showed that EP was converted from an ADP-sensitive to an ADP-insensitive form by a single turnover of the ATPase reaction. 1. After initiating the reaction by adding NaCl, almost all the EP was at first sensitive to added ADP, but its sensitivity to ADP decreased with increase in the time interval between the additions of NaCl and of ADP. 2. Both in the presence and absence of KCl, the time course of the replacement of ADP-sensitive EP by ADP-insensitive EP coincided with the time course of the decomposition of EP after addition of EDTA.     

Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. I. Exchange and hydrolysis kinetics at millimolar nucleotide concentrations

Parallel measurements in steady-state of ATP hydrolysis rate (vhydr) and the simultaneous reverse reaction, i.e., the ADP-ATP exchange rate (vexch), allowed the determination of a kinetic parameter, KE, containing only the four rate constants needed to characterize the enzyme intermediates involved in the sequence (Formula: see text). In order to compare the properties of these enzyme intermediates under different sets of conditions, KE was measured at varying K+ and Na+ concentrations in the presence of millimolar concentrations of ATP, ADP and MgATP, using an enzyme preparation that was partially purified from bovine brain. (1) In the presence of Na+ (150 mM), K+ (20-150 mM) was found to increase the exchange rate and decrease the ATP hydrolysis rate at steady-state. As a result, KE increased at increasing K+. However, the value of KE found by extrapolation to K+ = 0 was 7-times lower than the value actually measured in the absence of K+. This finding indicates that one of the intermediates, EATP or EP, or both, when formed in the presence of Na+ alone, are different from the corresponding intermediate(s) formed in the presence of Na+ + K+ (at millimolar substrate concentration). (2) In the presence of 150 mM K+, Na+ (5-30 mM) was found to increase the ADP/ATP exchange as well as the ATP hydrolysis rate at steady-state. The ratio of the two rates was constant. This finding, when interpreted in terms of KE, indicates that Na+ does not have to leave the enzyme for ATP release to be accelerated by K+ in the backward reaction. This also is in opposition to the usual versions of the Albers-Post model, which does not have simultaneous presence of Na+ and K+.     

Sodium and proton transport in Mycoplasma gallisepticum.

When washed cells of Mycoplasma gallisepticum were incubated at 37 degrees C in 250 mM 22NaCl, the intracellular Na+ increased, and the K+ decreased. The addition of glucose to these Na+-loaded cells caused Na+ efflux and K+ uptake (both ions moving against concentration gradients). This effect of glucose was blocked by the ATPase inhibitor dicyclohexylcarbodiimide, which prevents the generation of a proton motive force in these cells. In additional experiments, Na+ extrusion was studied by diluting the 22Na+-loaded cells into Na+-free media and following the loss of 22Na+ from the cells. Glucose stimulated 22Na+ extrusion in such cells by a dicyclohexylcarbodiimide-sensitive mechanism. Proton movement was studied by measuring the pH gradient across the cell membrane with the 9-aminoacridine fluorescence technique. Glucose addition to cells preincubated with cations other than Na+ resulted in cell alkalinization (which was prevented by dicyclohexylcarbodiimide). This observation is consistent with the operation of a proton-extruding ATPase. When glucose was added to Na+-loaded cells and diluted into Na+-free media, intracellular acidification was observed, followed several minutes later by a dicyclohexylcarbodiimide-sensitive alkalinization process. The initial acidification was probably due to the operation of an Na+-H+ antiport, since Na+ exit was occurring simultaneously with H+ entry. When Na+-loaded cells were diluted into Na+-containing media, the subsequent addition of glucose resulted in a weak acidification, presumably due to H+ entry in exchange for Na+ (driven by the ATPase) plus a continuous passive influx of Na+. All of the data presented are consistent with the combined operation of an ATP-driven proton pump and an Na+ -H+ exchange reaction.     

Immunochemical characterization of a functional site of (Na+,K+)-ATPase

Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Studies on (Na+ +K+) activated ATPase. XLI. Effects of N-ethylmaleimide on overall and partial reactions.

1. Preincubation with N-ethylmaleimide inhibits the overall activity of highly purified (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations of rabbit kidney outer medulla. 2. This inhibition is decreased by addition of ATP or 4-nitrophenylphosphate under non-phosphorylating conditions, and also by addition of ADP or adenylylimidodiphosphate. 3. N-ethylmaleimide treatment leads to inhibition of K+-stimulated 4-nitrophenylphosphatase activity, Na+-stimulated ATPase activity, and phosphorylation by ATP as well as by inorganic phosphate. These inhibitions strictly parallel that of the overal (Na+ +K+)-ATPase reaction. 4. N-ethylmaleimide lowers the number of sites which are phosphorylated by inorganic phosphate, without affecting the dissociation constant of the enzyme-phosphate complex. 5. N-ethylmaleimide does not affect the relative stimulation by ATP of the K+-stimulated 4-nitrophenylphosphatase activity. 6. These effects of N-ethylmaleimide can be explained as a complete loss of active enzyme, either by reaction of N-ethylmaleimide inside the active center, or by alterations in the quaternary structure through reactions outside the active center.     

Kinetic studies on the ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase. Evidence for the K.S.T. mechanism with two enzyme-ATP complexes and two phosphorylated intermediates of high-energy type.

The kinetic properties of the [3H]ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3.6.1,3] were investigated, using NaI-treated microsomes from bovine brain, and the following results were obtained. 1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45 micronM free Mg2+, 100 mMNaCl, 80 micronM ATP, and 160 micronM ADP at pH 6.5 and 4-5 degrees. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction parameters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 micronM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM. The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants Km, for both were 43 micronM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 micronM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 micronM ATP. 2. The N+-dependent [3H]ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+-concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H]ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration. From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP + ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP (formula: see text) produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step (formula: see text), determined by a method previously reported by us.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.     

Microsomal (Na- +K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors.

A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide.

In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na+ + K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or by p-nitrophenylphosphate. (ii) Protection by ATP of (Na+ + K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 micron Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (IV) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activities stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity.