Modulation of cardiac glycoside inhibition of (Na+ + K+)-ATPase by membrane lipids. Difference between species

The role of lipids in the modulation of the ouabain-sensitivity of membrane (Na+ + K+)-ATPase from different species has been studied using a reconstitution procedure which promotes lipid exchange during detergent depletion by Sephadex chromatography. Hybrid reconstitution of delipidated (Na+ + K+)-ATPase preparations from bovine brain into the lipids obtained from crab nerve enzyme preparations significantly reduces the sensitivity of the brain enzyme to inhibition by ouabain. Conversely, reconstitution of crab nerve enzyme into the lipids from bovine brain enzyme preparations increases the sensitivity of the crab enzyme to ouabain inhibition. These opposing effects demonstrate the role of membrane lipids in modulating the enzyme-inhibition relationship in preparations from these different species.     

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.     

Seasonal variations in the renal cortical (Na+ + K+)-ATPase and Mg2+-ATPase of a hibernator, the ground squirrel (Spermophilus richardsonii).

1. The specific activity of renal cortical (Na+ + K+)-ATPase of the Richardson ground squirrel is markedly reduced during hibernation, in contrast to the specific activity of the accompanying Mg2+-ATPase which is markedly increased. 2. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is unchanged by hibernation. 3. Both the non-linear thermal dependence of (Na+ + K+)-ATPase and the linear thermal dependence of Mg2+-ATPase are also unchanged by hibernation. 4. The energy of activation of both enzymes is unchanged during hibernation, or by comparison with that determined in awake controls. 5. There is no evidence for inherent "cold resistance" in these enzyme preparations compared to similar preparations from the non-hibernating rabbit. This parameter does not change during hibernation. 6. Both the rate and amount of specific [3H]-ouabain binding to the renal cortical preparations of (Na+ + K+)-ATPase decrease during hibernation. This decrease matches the fall in enzyme activity so that the ratio of pumping sites/unit of enzyme activity shows no seasonal variations. 7. These findings suggest that the amount of renal cortical (Na+ + K+)-ATPase enzyme falls during hibernation, but that the enzyme which remains functions with the same thermodynamic efficiency and identical biochemical characteristics of that found in the awake summer controls.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Influence of charge on the inactivation of membrane bound (Na+ + K+)-ATPase of Yoshida sarcoma cells by inhibitor proteins from cobra venom.

Inactivation of (Na+ + K+)-ATPase of Yoshida sarcoma cells and beef brain microsomes by phospholipase A2 and a cytotoxin P6 from snake venom has been examined in relation to their activity to degrade phospholipids. Cytotoxin P6 which was most basic and devoid of phospholipase activity was most effective in inhibiting the (Na+ + K+)-ATPase of Yoshida sarcoma cells. Phospholipase A2 from Naja naja which was most active in degrading phospholipids was least effective in inhibiting (Na+ + K+)-ATPase in Yoshida sarcoma cells or in beef brain microsomes. Addition of trace amounts of cytotoxin P6 to the phospholipase considerably enhanced the inactivation of (Na+ + K+)-ATPase. The evidence suggests that the charge of the inhibitor protein and its specific structure play an important role in the inactivation of (Na+ + K+)-ATPase.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Membrane (Na+ + K+)-ATPase of canine brain, heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures.

Effects of commonly used purification procedures on the yield and specific activity of (Na+ + K+)-ATPase (Mg2+-dependent, Na+ + K+-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na+ + K+)-ATPase and the ratio between (Na+ + K+)-ATPase and Mg2+-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na+ + K+)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na+ + K+)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzymes preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain.enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purfication procedure. These results indicate that the presentyl used purification procedures may alter the properties of membrane (Na+ + K+)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na+ + K+)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.     

Phosphorylation of two isozymes of (Na+ + K+)-ATPase by inorganic phosphate

The phosphorylation of two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase by 32Pi was studied under equilibrium conditions in various enzyme preparations from rat medulla oblongata, rat cerebral cortex, rat cerebellum, rat kidney, guinea pig kidney, and rabbit kidney. In ouabain-sensitive (Na+ + K+)-ATPases such as the brain, guinea pig kidney, and rabbit kidney enzymes, ouabain stimulated the Mg2+-dependent phosphorylation at lower concentrations, while a higher concentration was required for the stimulation of rat kidney (Na+ + K+)-ATPase, an ouabain-insensitive enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that two isozymes of the brain (Na+ + K+)-ATPase were also phosphorylated by 32Pi in the presence of ouabain. The properties of the phosphorylation were compared between the medullar oblongata (referred to as alpha(+] and the kidney (referred to as alpha) (Na+ + K+)-ATPases. The steady-state level of phosphorylation was achieved faster in the kidney enzymes than in the medulla oblongata enzyme. Phosphorylation without ouabain was greater in the kidney enzymes than in the brain enzymes. Furthermore, the former enzymes were inhibited by K+ much more than the latter. These findings suggest that the two isozymes of (Na+ + K+)-ATPase differ in their conformational changes during enzyme turnover.     

Inhibition of rat brain microsomal (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase by periodic acid

The effects of mild periodate exposure on the kinetics of (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase were studied using rat cerebral microsome preparations. Fifty percent inhibition of both enzyme activities was attained near 3 microM periodate concentrations. This inhibition was biphasic with time. Mg2+-ATPase and Mg2+-p-nitrophenylphosphatase activities were much less inhibited by periodate. Periodate inhibition was partially reversed by dimercaprol and dithiothreitol but not by diffusion. The possible reaction products formic acid, formaldehyde, glyceraldehyde, and acetaldehyde had no inhibitory effects in similar concentrations. Periodate exposure produced no detectable changes in the activation of (Na+ + K+)-ATPase by Na+, K+, Mg2+, or ATP. Residues shared by both (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase are both critical to hydrolytic function and sensitive to mild oxidation by periodate.     

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.     

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.     

Comparative temperature dependence of (Na+ + K+)-ATPase.

The temperature dependence of (Na+ + K+)-ATPase was measured, utilizing preparations of enzyme from heat and kidney of rats, hamsters, guinea pigs, ground squirrels, turtles, chickens, and ducks. The two hibernating species, hamsters and ground squirrels, were studied awake at normothermia and hibernating at 4 degrees C. The results for every species except the turtles showed the same temperature dependence established for (Na++K+)-ATPase from rabbit kidney with a quasi-linear dependence above 15 degrees C and little or no activity below 15 degrees C. Turtle enzymes showed a broad activity versus temperature curve with a fall-off at high and low temperatures. The data in all cases, including the turtle data, may be fitted by a previously described thermodynamic kinetic model. Further, the model will fith the turnover or decrease in enzyme activity at higher temperatures observed in a number of cases. These results do not support the widely imputed ion pumping role for (Na++K+)-ATPase.     

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors.

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

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.     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Reversible in activation of purified (Na+ + K+)-ATPase from human renal tissue by cyclic AMP-dependent protein kinase.

Human renal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations which exhibited a non-linear reaction rate, contained high levels of membrane-bound cyclic AMP-dependent protein kinase, while this latter activity was much less or absent in purified preparations. A non-linear reaction rate was observed in a purified preparation of (Na+ + K+)-ATPase by reconstituting the enzyme into lipid vesicles with cyclic AMP-dependent protein kinase. The addition of cyclic AMP to the ATPase assay of these lipid vesicles inactivated the (Na+ + K+)-ATPase. The cytoplasmic fraction of the cell contained a nondialyzable factor, which prevented (or reversed) the cyclic AMP-mediated inactivation of the enzyme.     

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Involvement of sulfhydryl groups in the inhibition of brain (Na+ + K+)-ATPase by pyrithiamin

Brain (Na+ + K+)-ATPase was protected by low concentrations of GSH from the inhibitory effect of pyrithiamin. The possible involvement of sulfhydryl groups in the inhibition was then studied by comparing the effect of pyrithiamin with that of N-ethylmaleimide on the enzyme. The treatment of rat brain (Na+ + K+)-ATPase with thesee inhibitors caused a significant decrease in reactivity of the enzyme to N-ethyl[3H]maleimide. N-Ethylmaleimide, like pyrithiamin, inhibited the partial reactions of (Na+ + K+)-ATPase system in parallel with the inhibition of the overall reaction. An SDS-polyacrylamide gel electrophoresis procedure indicated that pyrithiamin and N-ethylmaleimide inhibited Na+-dependent phosphorylation of the alpha(+) form of rat brain (Na+ + K+)-ATPase more than that of alpha, though the selectivity for the alpha(+) seemed to be higher with the former inhibitor than in the latter. The treatment also decreased sensitivity of the enzyme to ouabain inhibition. However, pyrithiamin- and N-ethylmaleimide-induced inactivations of the enzyme differed in the efficacy of GSH for protection and in the effect of the kind of ligands present during the reaction. Furthermore, pyrithiamin did not appear to interact directly with sulfhydryl groups, but caused the formation of disulfide in bovine brain (Na+ + K+)-ATPase. In contrast to N-ethylmaleimide, pyrithiamin did not affect the sulfhydryl-enzymes such as alcohol dehydrogenase and L-alanine dehydrogenase. It is concluded that pyrithiamin modifies the functional sulfhydryl groups of brain (Na+ + K+)-ATPase in a way different from N-ethylmaleimide and causes a structural change and inactivation of the enzyme.