Changes of sodium and ATP affinities of the cardiac (Na,K)-ATase during and after nitric oxide deficient hypertension

In the cardiovascular system, NO is involved in the regulation of a variety of functions. Inhibition of NO synthesis induces sustained hypertension. In several models of hypertension, elevation of intracellular sodium level was documented in cardiac tissue. To assess the molecular basis of disturbances in transmembraneous transport of Na⁺, we studied the response of cardiac (Na,K)-ATPase to NO-deficient hypertension induced in rats by NO-synthase inhibition with 40 mg/kg/day N^G-nitro-L-arginine methyl ester (L-NAME) for 4 four weeks. After 4-week administration of L-NAME, the systolic blood pressure (SBP) increased by 36%. Two weeks after terminating the treatment, the SBP recovered to control value. When activating the (Na,K)-ATPase with its substrate ATP, no changes in K_m and V_max values were observed in NO-deficient rats. During activation with Na⁺, the V_max remained unchanged, however the K_Na increased by 50%, indicating a profound decrease in the affinity of the Na⁺-binding site in NO-deficient rats. After recovery from hypertension, the activity of (Na,K)-ATPase increased, due to higher affinity of the ATP-binding site, as revealed from the lowered K_m value for ATP. The K_Na value for Na⁺ returned to control value. Inhibition of NO-synthase induced a reversible hypertension accompanied by depressed Na⁺-extrusion from cardiac cells as a consequence of deteriorated Na⁺-binding properties of the (Na,K)-ATPase. After recovery of blood pressure to control values, the extrusion of Na⁺ from cardiac cells was normalized, as revealed by restoration of the (Na,K)-ATPase activity. (Mol Cell Biochem 000: 000-000, 1999)     

Regulatory role of nitric oxide on the cardiac Na, K-ATPase in hypertension

The present study was focused on regulatory role of nitric oxide on functional properties of the cardiac Na, K-ATPase in three various animal models of hypertension: spontaneously hypertensive male rats (SHR) with increased activity of nitric oxide synthase (NOS) by 60 % (Sh1), SHR with decreased activity of NOS by 40 % (Sh2) and rats with hypertension induced by L-NAME (40 mg/kg/day) with depressed activity of NOS by 72 % (LN). Studying the utilization of energy substrate we observed higher Na, K-ATPase activity in the whole concentration range of ATP in Sh1 and decreased activity in Sh2 and LN. Evaluation of kinetic parameters revealed an increase of Vmax value by 37 % in Sh1 and decrease by 30 % in Sh2 and 17 % in LN. The KM value remained unchanged in Sh2 and LN, but was lower by 38 % in Sh1 indicating increased affinity of the ATP binding site, as compared to controls. During the activation with Na+ we observed increased Vmax by 64 % and increased KNa by 106 % in Sh1. In Sh2 we found decreased Vmax by 40 % and increased KNa by 38 %. In LN, the enzyme showed unchanged Vmax with increased KNa by 50 %. The above data indicate a positive role of increased activity of NOS in improved utilization of ATP as well as enhanced binding of Na+ by the cardiac Na, K-ATPase.     

Effect of the pyridoindole antioxidant stobadine on ATP-utilisation by renal Na,K-ATPase in rats with streptozotocin-induced diabetes

The effect of the pyridoindole antioxidant stobadine on diabetes-induced changes of Na,K-ATPase, especially those concerning the utilisation of its substrate ATP, was investigated. Sixteen weeks of streptozotocin-induced diabetes (single i.v. dose of streptozotocin; 55 mg/kg) was followed by decrease in the enzyme activity. This effect was emphasised in the presence of higher concentrations of substrate and in the presence of 8 mmol x l(-1) ATP it represented 20%. It might be a consequence of altered functional properties of Na,K-ATPase as suggested by 20% decrease in the V(max) value along with decrease in the K(m) value by 20%. Administration of 0.05% (w/w) stobadine in the diet to diabetic rats improved the function of renal Na,K-ATPase with respect to utilisation of ATP as suggested by significant increase in the enzyme activity in the whole concentration range of ATP investigated as a consequence of V(max) elevation to the level comparable to absolute controls. In conclusion, stobadine may play a positive role in restoring the functional properties of renal Na,K-ATPase, especially concerning the utilisation of energy derived from hydrolysis of ATP, improving thus the maintenance of ionic homeostasis during diabetes.     

Short-term NO synthase inhibition and the ATP affinity of cardiac Na,K-ATPase

It was previously shown that 4 hours lasting inhibition of nitric oxide synthesis by administration of an L-arginine analogue, the A(G)-nitro-L-arginine methyl ester (L-NAME) changed the affinity of the Na-binding site of Na,K-ATPase thus resulting in elevation of enzyme activity especially at higher concentrations of sodium. Using the same experimental model, we focused our attention in the present study to the question of binding of ATP to the enzyme molecule in the left ventricle (LV), ventricular septum (S) and the right ventricle (RV) of the dog heart. Activation of the enzyme by increasing concentrations of ATP revealed a significant increase of the Vmax only in septum (by 38 %). The K(M) increased significantly in septum (by 40 %) and in left ventricle (by 56 %) indicating an altered sensitivity of the ATP-binding site of Na,K-ATPase in the hearts of NO-deficient animals. The alterations of Na,K-ATPase in its ability to bind and hydrolyze ATP are localized to the tissue surrounding the cavity of the left ventricle.     

Gender difference in functional properties of Na,K-ATPase in the heart of spontaneously hypertensive rats

The aim of present study was the investigation of functional properties of the cardiac Na,K-ATPase in 16 weeks old male and female spontaneously hypertensive rats (SHR). The Na,K-ATPase activity in the presence of increasing concentrations of ATP, as well as Na(+) was lower in SHR of both genders, as compared to respective normotensive controls. Evaluation of kinetic parameters revealed a significant decrease of the maximum velocity (V(max)) in males (30% for ATP-activation, 40% for Na(+)-activation), as well as in females (24% for ATP, 29% for Na(+)), indicating a hypertension-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. Insignificant changes were observed in the value of Michaelis-Menten constant (K(m)) in both cases. The concentration of sodium that gives half-maximal reaction velocity (K(Na)), increased by 38% in male and by 70% in female SHR. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na,K-ATPase molecules are probably responsible for the deteriorated enzyme-function in hearts of SHR. Direct comparison of SHR of both genders showed, that the enzyme from female hearts seems to be adapted better to hypertension as documented by its increased activity as a consequence of improved ability to bind and utilize ATP, as suggested by 32% decrease of K(m) value in females. In addition, the enzyme from female hearts is able to increase its activity (by 41%) in the presence of increasing sodium concentration even in the range where the enzyme from male hearts is already saturated.     

Short-term NO synthase inhibition and the Na+-binding properties of cardiac Na,K-ATPase

It is known that hypertension is accompanied by increased [Na+]i. The functional properties of Na,K-ATPase, which transports the Na+ out and K+ into myocardial cells during the relaxation phase, were investigated in the left ventricle (LV), septum (SV) and the right ventricle (RV) of anesthetized dogs with moderate acute blood pressure elevation elicited by short-term (4-hour) NO synthase inhibition. The NO-insufficiency was induced by administration of an L-arginine analogue, the N(G)-nitro-L-arginine methyl ester (L-NAME). Concerning the function of Na,K-ATPase under the conditions of lowered NO synthesis, we focused our attention to the binding of Na+ to the enzyme molecule. Activation of the enzyme by increasing Na+ concentrations revealed significant changes in both the maximal velocity (Vmax) and the affinity for Na+ (K(Na)) in all investigated heart sections. The Vmax increased by 27% in LV, by 87% in SV and by 58% in RV. The K(Na) value increased by 86% in LV, by 105% in SV and by 93% in RV, indicating an apparent decrease in the sensitivity of the Na+-binding site in the Na,K-ATPase molecule. This apparently decreased pump affinity for Na+ together with the increase of Vmax suggest that, during the short-term inhibition of NO synthesis, the Na,K-ATPase is capable of extruding the excessive Na+ from the myocardial cells more effectively at higher [Na+]i, as compared to the Na,K-ATPase of control animals.     

Functional alterations of cardiac (Na,K)-ATPase in L-NAME induced hypertension

(Na,K)-ATPase, an enzyme involved in the active translocation of Na+ and K+ ions across cell membranes was shown to be affected by nitric oxide (NO) in various tissues. In the present study the functional alterations of (Na,K)-ATPase after chronic inhibition of nitric oxide synthesis were studied in rat hearts. Four weeks lasting administration of an L-arginine analogue, the N(G)-nitro-L-arginine methyl ester (L-NAME) induced an increase in the systolic blood pressure of about 36%. In this hypertension the kinetic parameters Km and Vmax for ATP-activation of the (Na,K)-ATPase did not show any significant changes. Activation of the enzyme by its cofactor Na+ revealed no change in the Vmax, but the K(Na) increased by 50%. Two weeks after terminating the administration of L-NAME the blood pressure returned to control values. In these conditions the activity of (Na,K)-ATPase increased, due to enlarged affinity of the ATP-binding site as revealed from the diminished Km value for ATP. The K(Na) value for activation with Na+ returned to control value. Our findings indicate that there is no change in energy utilization by the (Na,K)-ATPase during L-NAME induced hypertension in the heart. The transport properties of the enzyme are deteriorated, due to its decreased sensitivity to Na+. This inhibition of the (Na,K)-ATPase might be responsible for the increase of [Na+]i during lowered NO synthesis. In hearts from rats that recovered from the hypertension, the (Na,K)-ATPase increases its activity due to improved ATP binding properties.     

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.     

Sodium and ATP affinities of the cardiac (Na,K)-ATPase in relation to nitric oxide synthesis in spontaneously hypertensive rats

The (Na,K)-ATPase is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembraneous efflux of Na(+) from cardiac cells in spontaneously hypertensive rats (SHR) with increased synthesis of nitric oxide (NO). In the investigated group of SHR the systolic blood pressure was increased by 64% and the synthesis of NO was increased by 60% in the heart. When activating the cardiac (Na,K)-ATPase with substrate, its activity was higher in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed an increase of the V(max) (by 37%) probably due to increased affinity of the ATP-binding site as indicated by the lowered K(m) value (by 38%) in SHR. During activation with Na(+), we observed no change in the enzyme activity below 10 mmol/l of NaCl whereas in the presence of higher concentrations of NaCl the (Na,K)-ATPase was stimulated. The value of V(max) increased (by 64%), however the K(Na) increased (by 106%), indicating an adaptation of the Na(+)-binding site of the enzyme to increased [Na(+)](i). Thus the (Na,K)-ATPase in our SHR group is able to extrude the excessive Na(+) from myocardial cells more effectively also at higher [Na(+)](i), while the enzyme from controls is unable to increase its activity further. This improvement of the (Na,K)-ATPase function is supported also by increased affinity of its ATP-binding site probably due to enhanced NO-synthesis.     

Kinetics behaviors of Na,K-ATPase: comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme

We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C(12)E(8) solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na(+)-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K(0.5)=6 microM and 4 microM for reconstituted enzyme and purified form, respectively) and another of low affinity (K(0.5)=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (alphabeta)(2) oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg(2+) concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K(0.5) value of 0.4 mM. The Na(+) ions also presented a single saturation curve with V(M)=551 U/mg and K(0.5)=0.2 mM with cooperative effects. The activity was also stimulated by K(+) ions through a single curve of saturation sites (K(0.5)=2.8 mM), with cooperative effects and V(M)=641 U/mg. The lipid microenvironment close to the proteic structure and the K(+) internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.     

Gender specific influence of fish oil or atorvastatin on functional properties of renal Na,K-ATPase in healthy Wistar and hypertriglyceridemic rats

For better understanding of pathophysiological processes leading to increased retention of sodium as a consequence of hyperlipidemia, the properties of renal Na,K-ATPase, a key enzyme involved in maintaining sodium homeostasis in the organism, were studied. Enzyme kinetics of renal Na,K-ATPase were used for characterization of ATP- and Na(+)-binding sites after administration of fish oil (FO) (30 mg·day(-1)) or atorvastatin (0.5 mg·100 g(-1)·day(-1)) to healthy Wistar rats and rats with hereditary hypertriglyceridemia of both genders. Untreated healthy Wistar and also hypertriglyceridemic female rats revealed higher Na,K-ATPase activity as compared to respective untreated male groups. Hypertriglyceridemia itself was accompanied with higher Na,K-ATPase activity in both genders. Fish oil improved the enzyme affinity to ATP and Na(+), as indicated by lowered values of K(m) and K(Na) in Wistar female rats. In Wistar male rats FO deteriorated the enzyme in the vicinity of the Na(+)-binding site as revealed from the increased K(Na) value. In hypertriglyceridemic rats FO induced a significant effect only in females in the vicinity of the sodium binding sites resulting in improved affinity as documented by the lower value of K(Na). Atorvastatin aggravated the properties of Na,K-ATPase in both genders of Wistar rats. In hypertriglyceridemic rats protection of Na,K-ATPase was observed, but this effect was bound to females only. Both treatments protected renal Na,K-ATPase in a gender specific mode, resulting probably in improved extrusion of excessive intracellular sodium out of the cell affecting thus the retention of sodium in hHTG females only.     

The amino acid sequence 442GDASE446 in Na/K-ATPase is an important motif in forming the high and low affinity ATP binding pockets

A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the Na-dependent phosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km for p-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and its inhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was approximately 2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20 approximately 50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each KmP value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442GDASE446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg2+, Na+, and K+ effects.     

Binding of Na+ ions to the Na,K-ATPase increases the reactivity of an essential residue in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI), a new imidazole-based probe, results in irreversible loss of enzymatic activity. Inactivation of 95% of the Na,K-ATPase activity is achieved by the covalent binding of 1 molecule of [3H]NIPI to a single site on the alpha-subunit of the Na,K-ATPase. The reactivity of this site toward NIPI is about 10-fold greater when the enzyme is in the E1Na or sodium-bound form than when it is in the E2K or potassium-bound form. K+ ions prevent the enhanced reactivity associated with Na+ binding. Labeling and inactivation of the enzyme is prevented by the simultaneous presence of ATP or ADP (but not by AMP). The apparent affinity with which ATP prevents the inactivation by NIPI at pH 8.5 is increased from 30 to 3 microM by the presence of Na+ ions. This suggests that the affinity with which native enzyme binds ATP (or ADP) at this pH is enhanced by Na+ binding to the enzyme. Modification of the single sodium-responsive residue on the alpha-subunit of the Na,K-ATPase results in loss of high affinity ATP binding, without affecting phosphorylation from Pi. Modification with NIPI probably alters the adenosine binding region without affecting the region close to the phosphorylated carboxyl residue aspartate 369. Tightly bound (or occluded) Rb+ ions are not displaced by ATP (4 mM) in the inactivated enzyme. Thus modification of a single residue simultaneously blocks ATP acting with either high or low affinity on the Na,K-ATPase. These observations suggest that there is a single residue on the alpha-subunit (probably a lysine) which drastically alters its reactivity as Na+ binds to the enzyme. This lysine residue is essential for catalytic activity and is prevented from reacting with NIPI when ATP binds to the enzyme. Thus, the essential lysine residue involved may be part of the ATP binding domain of the Na,K-ATPase.     

Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.     

Erythrocyte ion transport and membrane lipid composition in young and adult rats with NO-deficient hypertension

The aim of our study was to search for abnormalities of sodium and potassium transport in erythrocytes of male Wistar rats subjected to chronic L-NAME treatment (40 mg/kg/day) for 4 weeks either from weaning (4-week-old) or in adulthood (12-week-old). Sodium content, Na(+),K(+)-pump and Na(+),K(+)-cotransport activity, cation leaks as well as membrane cholesterol and phospholipid contents were determined in fresh erythrocytes. Chronic inhibition of NO synthase elicited similar blood pressure rise in both age groups which did not differ in the degree of NO synthase inhibition. No significant ion transport abnormalities were disclosed in erythrocytes of young NO-deficient rats, whereas erythrocyte Na(+) content, outward Na(+),K(+)-cotransport and inward Na(+) leak were significantly reduced in adult hypertensive animals compared to age-matched controls. It should be noted that the erythrocytes of adult control rats were characterized by higher activity of Na(+),K(+)-pump and Na(+),K(+)-cotransport, increased Na(+) and Rb(+) leaks and elevated membrane cholesterol content compared to those of young normotensive controls. Increased Na(+) leak and elevated membrane cholesterol content but reduced membrane phospholipid content were revealed in erythrocytes of adult hypertensive rats when compared to young hypertensive rats. It can be concluded that young and adult Wistar rats did not differ in the extent of NO synthase inhibition and blood pressure rise elicited by chronic L-NAME treatment. Our results exclude the important participation of classical sodium transport abnormalities in the pathogenesis of this NO-deficient form of experimental hypertension.     

Nonenzymatic glycation of Na,K-ATPase. Effects on ATP hydrolysis and K+ occlusion

Glycation of the Na,K-ATPase in vitro (formation of Schiff base with glucose followed by reduction with NaCNBH3) shows the presence of three classes of reactive amino groups that differentially affect catalysis and cation binding. Reaction in the absence of ATP results in irreversible inhibition of enzyme activity with a t1/2 of 53 min. This is due to modification of one class of amino groups that affect the catalytic domain of the enzyme. In the presence of ATP, glycation first results in a shift in the steady state kinetics of ATP hydrolysis from substrate activation to Michaelis-Menten kinetics accompanied by an increase in the apparent affinity for K+ in the p-nitrophenylphosphatase reaction. This change in kinetic properties occurs with a t1/2 of 9 min and results in the complete loss of K+ occlusion. Incorporation of glucose is into the catalytic subunit, remote from the N-terminal end. Apparent total inhibition of K+ occlusion occurs with a stoichiometry 0.8 mol of glucose incorporated per mol of enzyme. Therefore, there is a rapidly reacting amino group that affects the cation binding domain of the Na,K-ATPase. More slowly, with a t1/2 of 9 h, the ATP hydrolysis kinetics change from Michaelis-Menten to substrate inhibition without recovery of K+ occlusion, showing that, in the E1 conformation, there is a third, slower reacting class of amino groups in the Na,K-ATPase that affects low affinity nucleotide interaction with the catalytic subunit.     

Kinetic parameters of Na,K-ATPase in the gills of crabs adapted to freshened sea water]

The dependence of Na,K-ATPase activity on concentrations of ATP, Na+, K+, Mg2+ and ouabain in the membrane preparations of crab gills was studied. The first group of crabs was adapted to freshened (25%) and the second one--to normal (100%) sea water. A 40-day adaptation of crabs to the freshened sea water results in an increase of maximal activity of Na,K-ATPase, but does not affect the enzyme affinity for ATP, Na+, K+, Mg2+ and ouabain, as well as its cooperative properties. It is assumed that adaptation of crabs to freshened sea water is accompanied by an accumulation of Na, K-ATPase in the epithelial cell membranes or crab gills without causing any qualitative changes of the enzyme.     

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.     

Sodium and ATP affinities of the cardiac Na(+),K(+)-ATPase in spontaneously hypertensive rats

The Na(+),K(+)-ATPase is postulated to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembrane Na(+)-efflux from cardiac cells in spontaneously hypertensive rats (SHR). In the investigated group of SHR the systolic blood pressure and the heart weight were increased by 48% and by 60%, respectively. Upon activating the cardiac Na(+),K(+)-ATPase with substrate, its activity was lower in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (Vmax) by 28% which was accompanied with lowered affinity of the ATP-binding site as indicated by the increased value of Michaelis-Menten constant (Km) by 354% in SHR. During activation with Na(+), we observed an inhibition of the enzyme in hearts from SHR at all tested Na(+) concentrations. The value of Vmax decreased by 37%, and the concentration of Na(+) that gives half maximal reaction velocity (KNa) increased by 98%. This impairment in the affinity of the Na(+)-binding site together with decreased affinity to ATP in the molecule of the Na(+),K(+)-ATPase are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of SHR.     

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.