Na+-ATPase in the gills of bass (Dicentrarchus labrax)

The authors evidence a Mg2+ dependent ATPase activity stimulated by Na+ in absence of K+ in bass gill microsomes. As this stimulated ATPase shows different features from "baseline" activity measured in the absence of both Na+ and K+ ions (Mg2+-ATPase) and from 1mM ouabain sensitive (Na+ + K+)-ATPase, it has been ascribed to a distinct Na+-ATPase. In the present paper the optimal conditions for bass gill Na+-ATPase assay and the temperature dependence of the enzyme are reported. Moreover the Na+-ATPase appears to be insensitive to 1mM ouabain and 100% inhibited by 2,5mM ethacrynic acid. It is suggested a parallel diffusion of Na+- and (Na+ + K+)-ATPase and a possible physiological role of Na+ATPase in osmoregulation.     

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.     

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.     

Free fatty acids and (Na+,K+)-ATPase: effects on cation regulation, enzyme conformation, and interactions with ethanol

Effects of free fatty acids on parameters of (Na+,K+)-ATPase regulation related to enzyme conformation were examined. Sensitivity to inhibition by free fatty acid increased as the number of double bonds increased. Free fatty acids reduced affinity for K+ or Na+ at their regulatory sites without altering apparent K+ affinity at its high-affinity site, and increased apparent affinity for ATP. The apparent E2/E1 ratio and apparent delta H and delta S for the E1-E2 transition were reduced by fatty acid. High K+ or low temperature reduced the sensitivity of enzyme to inhibition by free fatty acid. In the presence of low K+, arachidonic acid potentiated inhibition of phosphatase activity by ethanol. Arachidonic acid alone had little effect on the rate of ouabain binding, but accelerated ouabain binding in the presence of K+. These data suggest that fatty acids alter (Na+,K+)-ATPase by preventing the univalent cation-mediated transition to E2, the K+-sensitive form of enzyme. (Na+,K+)-ATPase could potentially be influenced in vivo by free fatty acids released by phospholipases or during hypoxia, or by changes in membrane lipid saturation.     

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.     

Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes.

The orientation of the enzyme Mg(2+)-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+, K+)-ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg(2+)-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external localization of the active site. Furthermore, the enzymatic activity resulted partially inhibited by treatment of the chicken TT vesicles with proteinase K or p-hydroxymercuribenzoate. Concanavalin A stimulated 4-fold the chicken TT Mg(2+)-ATPase activity, an effect not potentiated by detergent permeabilization of the intact vesicles, indicating that lectin-binding sites were also solvent accessible. This stimulatory effect was not observed in native or permeabilized rabbit TT vesicles. From these results we conclude that the TT Mg(2+)-ATPase is an ectoenzyme with its nucleotide-hydrolyzing site and glycosylated regions facing the extracellular space. Inhibitors of ion-motive ATPases did not modify the enzyme activity, suggesting a different physiological role for the TT Mg(2+)-ATPase which may be involved in the regulation of muscle fiber functions affected by extracellular ATP levels.     

Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg2+)-ATPase activity in islets of Langerhans

Studies were undertaken to determine whether factors which affect insulin secretion may exert their effects by altering the activity of an islet-cell plasma membrane Ca2+ extrusion pump. The insulin secretagogue, D-glucose, and a variety of phosphorylated hexoses, glucose 6-P, glucose 1,6-P, fructose 6-P, and fructose 2,6-P, were evaluated for their effect on an islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase and were found to be ineffective in altering enzyme activity. D-Glucose also did not alter the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Similarly, cAMP, the catalytic subunit of cAMP-dependent protein kinase, arachidonic acid, or prostaglandin E2 did not affect either the plasma membrane (Ca2+ + Mg2+)-ATPase or the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Whereas previous studies have suggested that D-glucose and/or cAMP may inhibit ATPase activities in islets, these results indicate that the agents, i.e., D-glucose and cAMP, which stimulate and/or potentiate insulin secretion from the islet cell, do not modify Ca2+ fluxes by directly regulating the islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase. In contrast, the acidic phospholipids, phosphatidic acid and phosphatidylserine, stimulated the enzyme activity in a concentration-dependent manner whereas phosphatidylcholine had only a minimal effect. The diacylglycerol, dilinolein, stimulated the (Ca2+ + Mg2+)-ATPase activity in the presence of phosphatidylserine, but not in the absence of phospholipids. These effects were independent of phospholipid-stimulated protein phosphorylation in the islet-cell plasma membrane under the conditions of the ATPase assay.     

Effect of Fatty Acids on [3H]Ouabain Binding to Cerebromicrovascular (Na++ K+)-ATPase

The effects of short- and long-chain fatty acids on the cerebromicrovascular (Na+ + K+)-ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37-110 micrograms) and was time-dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose-dependent manner, with a 105% increase over basal levels at 100 microM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4-Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 64.7 +/- 2.0 nM and a binding capacity (Bmax) of 10.1 +/- 1.5 pmol/mg protein. In the presence of 100 microM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 +/- 2.7 nM (p less than 0.01), whereas the Bmax remained virtually unchanged (12.5 +/- 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4-pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long-chain polyunsaturated fatty acids may be involved in the regulation of blood-brain barrier (Na+ + K+)-ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.     

Various properties of the Na+, K(+)-ATPase and the Mg (2+)-ATPase in erythrocytes from normotensive and hypertensive subjects]

In the present work we reported the results of the study of erythrocyte membrane Na+,K(+)-adenosine triphosphatase (ATPase) and Mg(2+)-ATPase in patients with essential hypertension and controls. In the 40 patients with hypertension, a more marked decrease of Na+, K(+)-ATPase was observed. The behavior of the enzyme at Mg2+ activation, ouabain inhibition and the response to different temperature suggest the possibility of differences between the two groups. The normal erythrocyte Mg(2+)-ATPase activity in two groups suggest also the possible role of ratio Na+, K(+)-ATPase/Mg(2+)-ATPase in the study of essential hypertension. However the relevance of magnesium and Mg(2+)-ATPase to the pathogenesis of essential hypertension remains unclear but merits further study. On the basis of these considerations the aim of the present study was to identify, in a kinetic approach, the presence of different abnormalities of Na+ transport and Na+, K(+)-ATPase in erythrocytes from patients with essential hypertension. Much evidence has supported the hypothesis that essential hypertension is a heterogeneous disease in the pathophysiological mechanisms as well as in its clinical and therapeutical consideration.     

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.     

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.     

Inhibition of duodenal enterocyte Mg2+-ATPase by arachidonic acid is not mediated by an effect on protein kinase C

Active absorption processes in the duodenal enterocyte are driven by various ATPases. It is known that the activity of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase can be modulated by polyunsaturated fatty acids of the n-6 series, for example by linoleic and gamma-linolenic acids. These effects may be achieved by protein phosphorylation via protein kinase C. The present study was undertaken to determine the effect of arachidonic acid on Mg2+-ATPase (measured colorimetrically) activity in basolateral membranes prepared from rat duodenum. It shows, for the first time, significant dose-dependent inhibition of Mg2+-ATPase (26-62%) by arachidonic acid (10-50 microg/ml) which already takes place after one minute of exposure, indicating involvement of a rapid signal transduction mechanism. Addition of the protein kinase C inhibitors bisimidolylmaleimide (2.5 microM) and calphostin (0.5 microM) did not influence the action of arachidonic acid on Mg2+-ATPase; protein kinase C involvement in this process is thus not indicated.     

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

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.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Effect of gamma-aminobutyric acid on Mg2+-ATPase of brain microsomes of the fish Abramis brama L.]

The influence of the GABA on the Mg(2+)-ATPase from microsomal fraction of fish brain (Abramis brama L.) was investigation. Preincubation of the microsomes with different concentration of GABA (10(-8)-10(-4) M) stimulated Mg(2+)-ATPase activity. This effect of neuromediator is sensitive to picrotoxine (10(-4) M). It was established that Mg(2+)-ATPase activity stimulated by anions (Cl > Br > F), inhibited by SCN- and not effected by HCO3-. The influence of the anions on the Mg(2+)-ATPase is liable to be inhibited by picrotoxine. It was supposed that anion-sensitive Mg(2+)-ATPase is associated functionally with GABAa-receptor.     

Effect of nitric oxides and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase activity in myometrium sarcolemma

The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane.     

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.