Sugar moiety of cardiac glycosides is essential for the inhibitory action on the palytoxin-induced K+ release from red blood cells

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.     

Effects of synthetic and naturally occurring flavonoids on Na+,K+-ATPase: aspects of the structure-activity relationship and action mechanism

A comparative study was made of the effects of 15 synthetic and naturally occurring flavonoids on the hydrolytic activity of Na+, K+-adenosine triphosphatase (ATPase). Twelve of the flavonoids examined were mono-hydroxy or mono-methoxy derivatives. All inhibited Na+, K+-ATPase from dog kidney cortex when present at concentrations from 40-1000 microM. Flavones possessing cyclohexyl instead of the phenyl group (i.e., 2-cyclohexyl-benzopyran-4-one derivatives), were the most potent with IC50 at 257-320 microM. Structure-activity relationships were observed among the following mono-substituted flavones as: i) 2-cyclohexyl-benzopyran-4-one much greater than 2-phenyl-benzopyran-4-one; ii) 2-cyclohexyl-7-hydroxybenzopyran-4-one greater than 2-cyclohexyl-6-hydroxybenzopyran-4-one greater than 2-cyclohexyl-5-hydroxybenzopyran-4-one. Some flavonoids showing potent inhibitory activity were also examined for ouabain-displacement activity on human erythrocytes. Hardly any of the flavonoids were able to block [3H]ouabain binding to erythrocytes. These results suggest that the mechanism by which flavonoid block Na+, K+-ATPase is not related to the cardiac glycoside-specific binding site(s) of this enzyme.     

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.     

Monovalent cation dependence of tissue plasminogen activator synthesis by HeLa cells

HeLa cells synthesize and secrete increased levels of tissue plasminogen activator (tPA) when incubated for 18 h with 10-20 nM phorbol myristate acetate. This response was inhibited by a number of conditions which affect intracellular Na+ and K+ concentrations. Removing extracellular Na+, while maintaining isotonicity with choline+, reduced the secretion of both functional and antigenic tPA in a linear fashion. A series of cardiac glycosides and related compounds strongly inhibited tPA secretion with the following rank order of potency: digitoxin = ouabain greater than digoxin greater than digitoxigenin greater than digoxigenin greater than digitoxose greater than digitonin. These compounds also inhibited cellular Na+/K+-ATPase activity over an identical concentration range. Two compounds which selectively increase cellular permeability to K+, valinomycin, and nigericin, strongly inhibited tPA secretion, with IC50 values of approximately 50 nM. In contrast, monensin, which selectively increases cellular permeability to Na+, was much less active. Valinomycin, but not nigericin, also inhibited cellular Na+/K+-ATPase activity. Phorbol myristate acetate, 5-20 nM, increased Na+/K+-ATPase activity up to 2-fold and tPA secretion up to 15-fold. We conclude that the secretion of tPA by HeLa cells treated with phorbol myristate acetate proceeds via a mechanism which requires extracellular Na+ and a functional Na+/K+-ATPase ("sodium pump") enzyme.     

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.     

Two active Na+/K+-ATPases of high affinity for ouabain in adult rat brain membranes

The degree of heterogeneity of active Na+/K(+)-ATPases has been investigated in terms of ouabain sensitivity. A mathematical analysis of the dose-response curves (inhibition of Na+/K(+)-ATPase) at equilibrium is consistent with the putative existence of three inhibitory states for ouabain two of high (very high plus high) and one of low affinity. The computed IC50 values are: 23.0 +/- 0.15 nM, 460 +/- 4.0 nM and 320 +/- 4.6 microM, respectively. The relative abundance of the three inhibitory states was estimated as: 39%, 36% and 20%, respectively. Direct measurements of [3H]ouabain-binding at equilibrium carried out on membrane preparations with ATP, Mg2+ and Na+ also revealed two distinct high affinity-binding sites, the apparent Kd values of which were 17.0 +/- 0.2 nM (very high) and 80 +/- 1 nM (high), respectively. Dissociation processes were studied at different ouabain concentrations according to both reversal of enzyme inhibition and [3H]ouabain release. The reversal of enzyme inhibition occurred at three different rates, depending upon the ouabain doses used (10 nM, 2 and 100 microM). When the high-affinity sites were involved (ouabain doses lower than 2 microM) the dissociation process was biphasic. A similar biphasic pattern was also detected by [3H]ouabain-release. The time-course of [3H]ouabain dissociation (0.1 microM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+/K(+)-ATPase alpha 1, alpha 2 and alpha 3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides.     

Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.     

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.     

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.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

Effects of glycyrrhizin and glycyrrhetinic acid on (Na+ + K+)-ATPase of renal basolateral membranes in vitro

Studies were made on the direct effects of glycyrrhizin and its aglycone, glycyrrhetinic acid on the activities of (Na+ + K+)-ATPase and (Ca2+ + Mg2+)-ATPase, a membrane bound Na+ and Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney. Glycyrrhetinic acid inhibited the activity of the Na+-pump enzyme dose-dependently (IC50 = 1.5 x 10(-4) M), but had no effect on that of the Ca2+-pump enzyme of kidney BLM and homogenates. Glycyrrhizin also inhibited the Na+-pump enzyme activity but had less effect (IC50 = 2 x 10(-3) M). The effects of these compounds were due to competitive inhibition with ATP binding to the enzyme (Ki = 12 microM) and so were different from that of ouabain, which inhibits the Na+-pump by binding to its extracellular K+-binding site. The direct effect of glycyrrhetinic acid on the membrane may be important role in the multiple actions of licorice.     

The calixarenes C-97 and C-107 stimulate influence of ouabain on the Na+,K+-ATPase activity in plasmatic membrane of smooth muscle cells

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the calix[4]arenes C-97 and C-107 (codes are shown) on ouabain effect on the Na+,K+-ATPase activity. It was shown that calixarenes in concentration 100 tiM inhibited by 97-98% the enzymatic Na+,K+-ATPase activity, while they did not practically influence on the basal Mg2+-ATPase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was < 0.1 microM while for ouabain it was 15-25 microM. The negative cooperative effect was typical of the inhibitory action of calixarenes, as well as ouabain: the value of Hills factor nH = 0.3-0.5 <1. The modelling compound M-3 (0.1 microM 4 microM)--a fragment of the calixarene C-107--did not practically influence the enzymatic activities as Na+,K+-ATPase and basal Mg2+-ATPase. Hence the influence of calixarene C-107 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments M-3 and effect of calixarene bowl, rather than by simple action of the fragment M-3. Calixarenes C-97 and C-107, used in concentration corresponding to values of I0.5 (40 and 60 nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-ATPase activity in the memrane fraction. Under coaction of ouabain with calixarene C-97 or C-107 there was no additive effect of the action of these inhibitors on the Na+,K+-ATPase activity. Calixarene C-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-ATPase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0 +/- 5.2 microM and 5.3 +/- 0.7 microM. It is concluded, that highly effective inhibitors of the Na+,K+-ATPase activity--calixarenes C-97 and C-107 can enhance the effect of the sodium pump conventional inhibitor--ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene C-97).     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Ouabain-binding site of (Na+ + K+)-ATPase in right-side-out vesicles has not an externally accessible SH group

The fluorescing sulfhydryl reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) inactivates purified (Na+ + K+)-ATPase at 20 microM. This inactivation results in a decrease of the ouabain-binding capacity of the enzyme. Treatment of (Na+ + K+)-ATPase, embedded in right-side-out-oriented vesicles, by DACM does not affect ouabain binding to the enzyme. Incorporation of DACM into the alpha subunit of (Na+ + K+)-ATPase embedded in right-side-out vesicles is also not affected by the presence or absence of 100 microM ouabain. It is therefore concluded that a sulfhydryl group does not reside within the ouabain-binding site of (Na+ + K+)-ATPase.     

Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.     

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.     

Characterization of a Mg2+-stabilized state of the (Na+ and K+)--stimulated adenosine triphosphatase using a fluorescent reporter group

A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an effect which was antagonized by both Na+ and ATP. Mg2+ also caused inhibition of K+-dependent dephosphorylation of the enzyme without inhibiting either (Na+)-ATPase activity or Na+-dependent phosphorylation. Mg2+ also induced a 5 to 6% enhancement in the fluorescence intensity of enzyme labeled with the fluorescent sulfhydryl reagent, 2-(4-maleimidylanilino)naphthalene-6-sulfonate. As in the case of Mg2+ inhibition of activity, the affinity for Mg2+ as an inducing agent for this effect was significantly reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced in magnitude by ouabain and prevented by oligomycin, specific inhibitors of the enzyme. In addition, K+ (and cations that substitute for K+ in supporting activity) induced a 3 to 4% enhancement in fluorescence intensity in the presence of Na+, Mg2+, and ATP, although the K+ and Mg2+ effects appeared to be different on the basis of their excitation spectra. The K+ effect was inhibited by ouabain and occurred with a rate greater than the rate of turnover of the enzyme, permitting its involvement in the catalytic cycle.     

(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.     

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.     

Long chain fatty acid inhibition of sodium plus potassium-activated adenosine triphosphatase from rat heart.

Long chain fatty acids were found to inhibit (Na+ + K+)-ATPase prepared from rat heart. Unsaturated and polyunsaturated fatty acids were more inhibitory than saturated fatty acids with myristic acid being the most inhibitory saturated fatty acid tested and linoleic the most inhibitory unsaturated fatty acid. As an example of fatty acid modification of the enzyme, inhibition of (Na+ + K+)-ATPase by oleate was examined. When compared to ouabain, inhibition of (Na+ + K+)-ATPase by oleate was found to be similar in that both were dependent on K+ concentration, but, in contrast to the almost instantaneous inhibition by ouabain, oleate inhibition was a slow process requiring over 20 min incubation at 37 degrees to produce maximum inhibition. Inhibition of rat heart (Na+ + K+)-ATPase by oleate was found to be readily reversible by washout. In the presence of albumin an oleate/albumin molar ratio greater than 7.5 was required for inhibition to occur. The activity of rat heart (Na+ + K+)-ATPase had a temperature optimum above 40 degrees and a discontinuous Arrhenius' plot with a transition temperature of 25 degrees. In the presence of oleate, however, the enzyme's optimum temperature decreased to below 40 degrees, the activation energy of the reaction at temperatures below 25 degrees was lowered from 24.7 kcal/mol to 12.6 kcal/mol and the enzyme had a linear Arrhenius' plot. The possibility of in vivo inhibition of cardiac (Na+ + K+)-ATPase under conditions of elevated fatty acids is discussed.