Simplified digitalis-like compounds acting on Na(+), K(+)-ATPase

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na(+),K(+)-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5beta,14beta-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na(+), K(+)-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.     

Effect of Na(+), K(+)-pump inhibitors on the sensory ganglia neurite growth

Inhibitors of Na(+), K(+)-pump belonging to the class of cardiac glycosides were investigated in organotypic tissue culture of dorsal root ganglia cells of 10-12 days old chicken embryos. The data obtained show that the application of cardiac glycosides (strophantin K and digoxin) in a wide range of concentrations controls the neurite growth in sensory neurons in the dose-dependent manner. It was shown, that at the concentrations of cardiac glycoside exceeding 1 x 10(-6) M the growth of neurites was totally inhibited. Our data indicate that cardiac glycoside have the down-regulation effect on the neurite growth. The data obtained indicate that the Na(+), K(+)-ATPase is involved in the control of the process of neurite growth as a signal transducer.     

Kinetics of K(+) occlusion by the phosphoenzyme of the Na(+),K(+)-ATPase

Investigations of K⁺-occlusion by the phosphoenzyme of Na⁺,K⁺-ATPase from shark rectal gland and pig kidney by stopped-flow fluorimetry reveal major differences in the kinetics of the two enzymes. In the case of the pig enzyme, a single K⁺-occlusion step could be resolved with a rate constant of 342 (±26) s⁻¹. However, in the case of the shark enzyme, two consecutive K⁺-occlusions were detected with rate constants of 391 (±19) s⁻¹ and 48 (±2) s⁻¹ at 24°C and pH 7.4. A conformational change of the phosphoenzyme associated with K⁺-occlusion is, thus, the major rate-determining step of the shark enzyme under saturating concentrations of all substrates, whereas for the pig enzyme the major rate-determining step under the same conditions is the E2 → E1 transition and its associated K⁺ deocclusion and release to the cytoplasm. The differences in rate constants of the K⁺ occlusion reactions of the two enzymes are paralleled by compensating changes to the rate constant for the E2 → E1 transition, which explains why the differences in the enzymes' kinetic behaviors have not previously been identified.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.     

Rate limitation of the Na(+),K(+)-ATPase pump cycle

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.     

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.     

Effects of Mg(2+) on activation of the (Na(+) + K(+)-dependent ATPase by Na(+1).

The effects of MgCl₂ on Na activation of three different enzymatic reactions catalyzed by a rat brain (Na + K)-dependent ATPase (adenosine 5′-triphosphatase) were studied. For the Na⁺-dependent ATPase reaction measured with 6 μm ATP, the K_0.5 for Na increased from 0.4 to 1.7 mm as the MgCl₂ concentration was raised from 50 to 2000 μm; the half-maximal effect occurred at a free Mg²⁺ concentration near 0.8 mm. By contrast, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na was again 0.4 mm, but further addition of 2 mm MgCl₂ then had little effect on the K_0.5 for Na. For the Na-dependent phosphorylation of the enzyme, measured with 6 μm ATP, the K_0.5 for Na increased similarly, from 0.2 to 0.8 mM, as the MgCl₂ concentration was raised from 50 to 2000 μm, but for the (Na + K)-dependent ATPase reaction the K_0.5 for Na was 13 mm and increased by only one-third as the MgCl₂ concentration was raised. The K_0.5 for K was also little affected by changes in MgCl₂ concentration. Finally, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na in the (Na + K)-dependent ATPase reaction decreased to 5 mm. These observations are considered in terms of an enzyme having high-affinity and low-affinity substrate sites, with occupancy of the low-affinity sites modifying Na activation differently, depending both on the specific reaction catalyzed and on whether occupancy is by free Mg²⁺ or by Mg-ATP.     

Kinetic Comparisons of Heart and Kidney Na(+),K(+)-ATPases

Most kinetic measurements of the partial reactions of Na⁺,K⁺-ATPase have been conducted on enzyme from mammalian kidney. Here we present a kinetic model that is based on the available equilibrium and kinetic parameters of purified kidney enzyme, and allows predictions of its steady-state turnover and pump current in intact cells as a function of ion and ATP concentrations and the membrane voltage. Using this model, we calculated the expected dependence of the pump current on voltage and extracellular Na⁺ concentration. The simulations indicate a lower voltage dependence at negative potentials of the kidney enzyme in comparison with heart muscle Na⁺,K⁺-ATPase, in agreement with experimental results. The voltage dependence is enhanced at high extracellular Na⁺ concentrations. This effect can be explained by a voltage-dependent depopulation of extracellular K⁺ ion binding sites on the E2P state and an increase in the proportion of enzyme in the E1P(Na⁺)₃ state in the steady state. This causes a decrease in the effective rate constant for occlusion of K⁺ by the E2P state and hence a drop in turnover. Around a membrane potential of zero, negligible voltage dependence is observed because the voltage-independent E2(K⁺)₂ → E1 + 2K⁺ transition is the major rate-determining step.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway

We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.     

Fluoride and beryllium interact with the (Na + K)-dependent ATPase as analogs of phosphate

Fluoride irreversibly inhibits the (Na + K)-ATPase, and this inactivation requires divalent cations (Mg²⁺, Mn²⁺, or Ca²⁺), is augmented by K⁺, but is diminished by Na⁺ and by ATP. Prior incubation with the aluminum chelator deferoxamine markedly slows inactivation, whereas adding 1 µM AlCl₃ speeds it, consistent with AlF _– ⁴ being the active species. Prior incubation of the enzyme with vanadate also blocks inactivation by fluoride added subsequently. Fluoride stimulates ouabain binding to the enzyme, and thus the analogy between AlF _– ⁴ and both orthophosphate and orthovanadate is reflected not only in the similar dependence on specific ligands for their enzyme interactions and their apparent competition for the same sites, but also in their common ability to promote ouabain binding. Beryllium also irreversibly inhibits the enzyme, and this inactivation again requires divalent cations, is augmented by K⁺, but is diminished by Na⁺ and by ATP. Similarly, prior incubation of the enzyme with vanadate blocks inactivation by beryllium added subsequently. Inactivation by beryllium, however, does not require a halide, and, unlike inactivation by fluoride, increases at basic pHs. These observations suggest that beryllium, as beryllium hydroxide complexes, acts as a phosphate analog, similar to AlF _– ⁴ and vanadate.Abbreviations EDTA ethylenediaminetetraacetate - EGTA Ethyleneglycol-bis(-aminoethylether)-N,N-tetraacetate - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)1,4-piperazine diethanesulfonic acid     

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Effect of catecholamines on activity of Na(+), K(+)-ATPase in neonatal piglet brain during posthypoxic reoxygenation

The present study examined the possible role of dopamine on the response of Na(+), K(+)-ATPase activity in the striatum of newborn piglets to 1 h of bilateral carotid ligation with hemorrhage and 2 h of recovery. Newborn piglets, 2-4 days of age and with and without prior treatment with alpha-methyl-p-tyrosine (AMT), an inhibitor of catecholamines synthesis, were used for the study. The oxygen pressure in the microvasculature of the cortex (PcO(2)) was measured by oxygen dependent quenching of the phosphorescence. In sham-operated animals the PcO(2) was 50+/-3 torr. Following ligation and hemorrhage the PcO(2) decreased to 8+/-0.5 torr. After release of ligation and reperfusion PcO(2) increased to 45+/-4 torr, a value not significantly different from controls, in approximately 30 min. There were no significant differences in PcO(2) between AMT treated and untreated animals. In sham-operated animals striatal Na(+),K(+)-ATPase was 29.1+/-3 micromol/mg protein per h and decreased by 25% after 2 h of recovery. Depleting the brain of catecholamines prior to ligation and hemorrhage abolished this decrease. It is postulated that the decrease in the level of dopamine in the brain prior to ligation and hemorrhage can be at least partly responsible for the observed decrease in activity of Na(+), K(+)-ATPase in the striatum of newborn piglets.     

Transport ATPase: thimerosal inhibits the Na+ +K+-dependent ATPase activity without diminishing the Na+-dependent ATPase activity.

A guinea pig kidney membrane preparation was incubated with thimerosal and then thoroughly washed. Comparison of the properties of the native and the modified membranes showed that (a) Na⁺+K⁺-dependent activity is substantially inhibited by thimerosal; (b) thimerosal does not diminish Na⁺-dependent ATPase activity; and (c) the thimerosal treated enzyme, like the native enzyme, is phosphorylated in the presence of Na⁺ and ATP, and dephosphorylated upon the addition of K⁺. It is suggested that thimerosal does not affect the binding of ATP to the high-affinity catalytic site, but that it blocks the binding of ATP to a low affinity modifying site the occupation of which is essential for the dissociation of the stable K⁺-dephosphoenzyme and the recycling of the enzyme.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Effect of the pyridoindole antioxidant stobadine on the cardiac Na(+),K(+)-ATPase in rats with streptozotocin-induced diabetes

In the present study we examined the effect of dietary supplementation with the pyridoindole antioxidant stobadine on functional properties of the cardiac Na(+),K(+)-ATPase in diabetic rats. Diabetes lasting sixteen weeks which was induced by a single i.v. dose of streptozotocin (55 mg x kg(-1)) was followed by decrease in the enzyme activity. Evaluation of kinetic parameters revealed a statistically significant decrease in the maximum velocity (Vmax) (32% for ATP-activation, 33% for Na(+)-activation), indicating a diabetes-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. The ATP-binding properties of the enzyme were not affected by diabetes as suggested by statistically insignificant changes in the value of Michaelis-Menten constant, K(M (ATP)). On the other hand, the affinity to sodium decreased as suggested by 54% increase in the K(M (Na+)) value. 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 diabetic animals. Administration of stobadine to diabetic rats dramatically improved the function of cardiac Na(+),K(+)-ATPase with regard to Na(+)-handling, as documented by statistically significant elevation of Vmax by 66 and 47% decrease in K(M (Na+)). Our data suggest that stobadine may prevent the diabetes-induced deterioration of cardiac Na(+),K(+)-ATPase, thus enabling to preserve its normal function in regulation of intracellular homeostasis of Na(+) and K(+) ions.     

Mechanism of the rate-determining step of the Na(+),K(+)-ATPase pump cycle

The kinetics of the E(2) --> E(1) conformational change of unphosphorylated Na(+),K(+)-ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E(2) conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/tau, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 +/- 3 s(-1) in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na(+) to the enzyme in the E(2) state with a dissociation constant of 31 +/- 7 mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/tau for both the NaCl and choline chloride titrations. The results suggest that Na(+) ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E(2) --> E(1) conformational transition by interaction with the E(2) state.     

The Occlusion of Rb(+) in the Na(+)/K(+)-ATPase. II. The effects of Rb(+), Na(+), Mg2(+), or ATP on the equilibrium between free and occluded Rb(+).

We used the direct route of occlusion to study the equilibrium between free and occluded Rb(+) in the Na(+)/K(+)-ATPase, in media with different concentrations of ATP, Mg(2+), or Na(+). An empirical equation, with the restrictions imposed by the stoichiometry of ligand binding was fitted to the data. This allowed us to identify which states of the enzyme were present in each condition and to work out the schemes and equations that describe the equilibria between the ATPase, Rb(+), and ATP, Mg(2+), or Na(+). These equations were fitted to the corresponding experimental data to find out the values of the equilibrium constants of the reactions connecting the different enzyme states. The three ligands decreased the apparent affinity for Rb(+) occlusion without affecting the occlusion capacity. With [ATP] tending to infinity, enzyme species with one or two occluded Rb(+) seem to be present and full occlusion seems to occur in enzymes saturated with the nucleotide. In contrast, when either [Mg(2+)] or [Na(+)] tended to infinity no occlusion was detectable. Both Mg(2+) and Na(+) are displaced by Rb(+) through a process that seems to need the binding and occlusion of two Rb(+), which suggests that in these conditions Rb(+) occlusion regains the stoichiometry of the physiological operation of the Na(+) pump.