Sensitivity of the (Na+ + K+)-ATPase to state-dependent inhibitors: Effects of digitonin and triton X-100

Treatment of a purified (Na⁺ + K⁺)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na⁺ + K⁺)-ATPase reaction inhibited more than the K⁺-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na⁺ + K⁺)-ATPase reaction but not the K⁺-phosphatase reaction; however, treatment with digitonin made the K⁺-phosphatase reaction almost as sensitive to oligomycin as the (Na⁺ + K⁺)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K⁺-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K⁺-phosphatase reaction more than the (Na⁺ + K⁺)-ATPase reaction). Both digitonin and Triton markedly increased the K_0.5 for K⁺ as activator of the K⁺-phosphatase reaction, with little effect on the K_0.5 for K⁺ as activator of the (Na⁺ + K⁺)-ATPase reaction. In contrast, increasing the K_0.5 for K⁺ in the K⁺-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K⁺-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E₁ conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E₂ state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K_0.5 for K⁺ in the K⁺-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na⁺ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na⁺ plus nucleotide.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ +K+)-dependent ATPase

Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100.     

(Na+ + K+)-ATPase: effects of detergents on the cross-linking of subunits in the presence of Cu2+ and o-phenanthroline.

When (Na+ + K+)-ATPase is reacted with Cu2+ or Cu2+-phenanthroline, cross-linking of the two subunits (alpha and beta) occurs. The major products are alpha,beta- and alpha,alpha-dimers. The alpha,beta-dimer is unstable in the presence of EDTA, but becomes stable when it is first exposed to digitonin or Triton X-100. Conversion of alpha-CU2+-beta to alpha-S-S-beta is suggested. If the enzyme that is pretreated with these detergents is used, only the stable alpha,beta-dimer is obtained, and the formation of alpha,alpha-dimer is inhibited. The data are consistent with alpha 2 beta 2 quaternary structure of the enzyme.     

The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase.

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.     

Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase

A dog kidney (Na+ + K+)-ATPase preparation also catalyzes K+-independent and K+-activated phosphatase reactions with p-nitrophenyl phosphate as substrate. K+-independent activity increases with declining pH over the range 7.5 to 5.8, whereas the other two activities decrease. The increased K+-independent activity is similar with imidazole, histidine, and several Good buffers, and is thus attributable to free H+, probably by affecting enzyme conformations rather than by changing affinity for Mg2+ or substrate or by H+ occupying specific K+-sites. The decrease in K+-phosphatase and (Na+ + K+)-ATPase activities with pH also occurs similarly with those buffers, and is not due to changes in apparent affinity for substrate or for cation activators. However, the Good buffers Pipes and ADA inhibit the K+-independent phosphatase reaction strongly, the K+-activated reaction moderately, and the (Na+ + K+)-ATPase reaction little; both contain two acidic groups, unlike the other buffers tested. Inhibition of the phosphatase reaction by Pipes is associated with a decreased apparent affinity for K+ and an increased sensitivity to inhibition by Na+ and ADP, consistent with Pipes hindering conformational transitions to the E2 enzyme forms required for phosphatase hydrolytic activity.     

Inhibition by vanadate of the reactions catalyzed by the (Na+ + K+)-stimulated ATPase. A transient state kinetic characterization

The interaction of vanadate with the (Na+ + K+)-stimulated ATPase from electric organ was investigated using the acid quench-flow technique. At 21 degrees C, incubation of the enzyme with 1.3 to 1.6 muM vanadate in the presence of 75 mM Na+ and 25 mM K+ strongly inhibits phosphorylation by ATP. Enzyme activity remaining under these conditions shows no change in the apparent rates of phosphorylation or dephosphorylation, although effects were noted which suggest that vanadate increases the reverse rate of dephosphorylation. Ten micromolar vanadate, sufficient to inhibit the (Na+ + K+)-stimulated ATPase by more than 98%, has no effect on phosphorylation in the presence of Na+ alone. Phosphoenzyme formed in the presence of Na+ and K+ consists of rapidly and slowly decaying components which differ in sensitivity to vanadate. Up to 2 muM vanadate suppresses predominantly the rapidly decaying phosphoenzyme, while at higher concentrations vanadate inhibits both the rate and level of formation of the slowly decaying phosphoenzyme. These results indicate that vanadate is a useful reagent for distinguishing between these two phosphorylation reactions.     

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.     

Studies on ouabain-complexed (Na+ +K+)-ATPase carried out with vanadate

Vanadate is able to promote the binding of ouabain to (Na+ +K+)-ATPase and it is shown that vanadate is trapped in the enzyme-ouabain complex. Also ouabain-bound enzyme, the formation of which was facilitated by (Mg2+ +Na+ +ATP) or (Mg2+ +Pi), is accessible to vanadate when washed free of competing ligands used for the promotion of ouabain binding. For vanadate binding to (Na+ +K+)-ATPase and to enzyme-ouabain complexes a divalent cation (Mg2+ or Mn2+) is indispensable, indicating that the cation does not remain attached to the ouabain-bound enzyme. K+ further increases vanadate binding in the absence of ouabain, but seems to have no additional role in case of vanadate binding to enzyme-ouabain complexes. Mn2+ is more efficient than Mg2+ in promoting binding of vanadate and ouabain to (Na+ +K+)-ATPase. That K+ in combination with Mn2+, in analogy with the effect in combination with Mg2+, increases the equilibrium binding level of vanadate and decreases that of ouabain does not seem to favour the hypothesis of selection of a special E2-subconformation by Mn2+. The vanadate-trapped enzyme-ouabain complex was examined for simultaneous nucleotide binding which could demonstrate a two-substrate mechanism per functional unit of the enzyme. The acceleration by (Na+ +ATP) of ouabain release from the (Mg2+ +Pi)-facilitated enzyme-ouabain complex does not, as anticipated, support such a mechanism. On the other hand, the deceleration of vanadate release as well as of ouabain release from a (Mg2+ +vanadate)-promoted complex could be consistent with a two-substrate mechanism working out-of-phase.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Fluorescent labeling of (Na+ + K+)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na+ + K+)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K+-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na+, K+, Mg2+, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na+, and Mg2+, and decreased by K+. These fluorescence changes likely reflect ligand-induced stabilization of the E1 or E2 states of the enzyme.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme

Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.     

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.     

Facilitation of ouabain binding to (Na+ + K+)-ATPase by vanadate at in vivo concentrations.

In the presence of Mg2+ vanadate was shown to facilitate ouabain binding to (Na+ + K+)-ATPase in much the same way as Pi does. Thus the hypothesis that vanadate interacts with the phosphate site of the enzyme seems to be supported by ouabain binding experiments. At given ouabain concentrations maximum binding is achieved at microM concentrations of vanadate whereas mM concentrations of Pi are needed. Na+ as well as K+ counteract ouabain binding but some cardiac glycoside binding is still possible at in vivo concentrations of these cations. A minor contamination of the enzyme preparations with vanadate could explain the in vitro binding of ouabain that can be obtained with Mg2+ and in the absence of Pi.     

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

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

Kinetic properties of C12E8-solubilized (Na+ + K+)-ATPase

The properties of the rectal gland (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether ( C12E8 ) have been investigated. The kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that Km for ATP for the (Na+ + K+)-ATPase is about 30 microM for the solubilized enzyme and about 100 microM for the membrane-bound enzyme. The Na+-form (E1) and the K+-form (E2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and eosin fluorescence responses to Na+ and K+. The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95-100% pure supernatant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C12E8 /protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C12E8 is required both for solubilization and inactivation. It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C12E8 , and its advantages in the assay of detergent-solubilized (Na+ + K+)-ATPase are discussed. The results show that (Na+ + K+)-ATPase can be solubilized in C12E8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C12E8 -solubilized (Na+ + K+)-ATPase is therefore a good model system for a solubilized membrane protein.     

Difference between neuronal and nonneuronal (Na+ + K+)-ATPases in their conformational equilibrium

Several experiments were carried out to study the difference between two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase in the conformational equilibrium. Rat brain (Na+ + K+)-ATPase was much more thermolabile than the kidney enzyme. Both enzymes were protected from heat inactivation not only by Na+ and K+, but also by choline in varying degrees, though there was a difference between the two enzymes in the protection by the ligands. The brain enzyme was partially protected from N-ethylmaleimide (NEM) inactivation by both Na+ and K+, but the effects of the ligands on NEM inactivation of the kidney enzyme were more complex. Though ligands differentially affected the thermostability and NEM sensitivity of the two enzymes, the effects were not simply related to the conformational states. The sensitivity of phosphoenzyme (EP) formed in the presence of ATP, Na+, and Mg2+ to ADP or K+ and K+-p-nitrophenyl phosphatase (pNPPase) was then studied as a probe of the differences in the conformational equilibrium between the two isozymes. The EP of the brain enzyme was partially sensitive to ADP, while those of the heart and kidney enzymes were not. At physiological Na+ concentrations the percentages of E1P formed by the brain and kidney enzymes were determined to be about 40-50 and 10-20% of the total EP, respectively. The hydrolytic activity of pNPP in the presence of Li+, a selective activator at catalytic sites of the reaction, was much higher in the kidney enzyme than in the brain enzyme. The inhibition of K+-stimulated pNPPase by ATP and Na+ was greater in the latter enzyme than in the former. These results suggest that neuronal and nonneuronal (Na+ + K+)-ATPases differ in their conformational equilibrium: the E1 or E1P may be more stable in the alpha(+) than in the alpha during the turnover, and conversely the E2 or E2P may be more stable in the latter than in the former.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: vanadate nad a dithioerythritol-dependent inhibitor.

A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.