Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.     

Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.     

The role of Mg2+ and K+ in the phosphorylation of Na+,K(+)-ATPase by ATP in the presence of dimethylsulfoxide but in the absence of Na+.

We have previously demonstrated that Na+,K(+)-ATPase can be phosphorylated by 100 microM ATP and 5 mM Mg2+ and in the absence of Na+, provided that 40% dimethylsulfoxide (Me2SO) is present. Phosphorylation was stimulated by K+ up to a steady-state level of about 50% of Etot (Barrabin et al. (1990) Biochim. Biophys. Acta 1023, 266-273). Here we describe the time-course of phosphointermediate (EP) formation and of dephosphorylation of EP at concentrations of Mg2+ from 0.1 to 5000 microM and of K+ from 0.01 to 100 mM. The results were simulated by a simplified version of the commonly accepted Albers-Post model, i.e. a 3-step reaction scheme with a phosphorylation, a dephosphorylation and an isomerization/deocclusion step. Furthermore it was necessary to include an a priori, Mg(2+)- and K(+)-independent, equilibration between two enzyme forms, only one of which (constituting 14% of Etot) reacted directly with ATP. The role of Mg(2+) was two-fold: At low Mg2+, phosphorylation was stimulated by Mg2+ due to formation of the substrate MgATP, whereas at higher concentrations it acted as an inhibitor at all three steps. The affinity for the inhibitory Mg(2+)-binding was increased several-fold, relative to that in aqueous media, by dimethylsulfoxide. K+ stimulated dephosphorylation at all Mg(2+)-concentrations, but at high, inhibitory [Mg2+], K+ also stimulated the phosphorylation reaction, increasing both the rate coefficient and the steady-state level of EP. Generally, the presence of Me2SO seems to inhibit the dephosphorylation step, the isomerization/deocclusion step, and to a lesser extent (if at all) the phosphorylation reaction, and we discuss whether this reflects that Me2SO stabilizes occluded conformations of the enzyme even in the absence of monovalent cations. The results confirm and elucidate the stimulating effect of K+ on EP formation from ATP in the absence of Na+, but they leave open the question of the molecular mechanism by which Me2SO, inhibitory Mg2+ and stimulating K+ interact with the Na+,K(+)-ATPase.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.     

Pb2+ and imidazole-activated phosphorylation by ATP of (Na+ + K+)-ATPase

In order to study whether Pb2+ and imidazole increase the ATP phosphorylation level of (Na+ + K+)-ATPase by the same mechanism, the effects of both compounds on phosphorylation and dephosphorylation reactions of the enzyme have been studied. Imidazole in the presence of Mg2+ increases steady-state phosphorylation of (Na+ + K+)-ATPase by decreasing, in a competitive way, the K+-sensitivity of the formed phospho-enzyme (E-P . Mg). If Pb2+ is present during phosphorylation, the rate of phosphorylation increases and a K+- and ADP-insensitive phosphointermediate (E-P . Pb) is formed. Pb2+ has no effect on the K+-sensitivity of E-P . Mg and EDTA is unable to affect the K+-insensitivity of E-P . Pb. These effects indicate that Pb2+ acts before or during phosphorylation with the enzyme. Binding of Na+ to E-P . Pb does not restore K+-sensitivity either. However, increasing Na+ during phosphorylation in the presence of Pb2+ leads to formation of the K+-sensitive intermediate (E-P . Mg), indicating that E-P . Pb is formed via a side path of the Albers-Post scheme. ATP and ADP decrease the dephosphorylation rate of both E-P . Mg and E-P . Pb. Above optimal concentration, Pb2+ also decreases the steady-state phosphorylation level both in the absence and in the presence of Na+. This inhibitory effect of Pb2+ is antagonized by Mg2+.     

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.     

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes

The present paper characterizes the Na+-stimulated ATPase activity present in basal-lateral plasma membranes from guinea-pig kidney proximal tubular cells. These characteristics are compared with those of the (Na+ + K+)-stimulated ATPase activity, and they are: (A) Na+-ATPase activity: (1) requires Mg2+; (2) may be activated by mu molar quantities of Ca2+; (3) optimal ratio Mg:ATP = 5:1-2 and Ka for Mg:ATP = 3:0.60 mM; (4) Ka for Na+:8 mM; (5) does not require K+; (6) is only stimulated by Na+ and Li+ (in a lower extent); (7) is similarly stimulated by the Na+ salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47 degrees C; (10) optimal pH: 6.9; (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na+ + K+)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP = 1.25:1 and Ka for Mg:ATP = 0.50: 0.40 mM; (4) Ka for Na+: 14 mM (data not shown); (5) needs K+ together with Na+; (6) K+ may be substituted by: Rb+ greater than NH+4 greater than Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, ITP, UTP, ADP, CTP; (9) optimal temperature: 52 degrees C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.     

Ethylenediamine as active site probe for Na+/K+-ATPase

(1) Ethylenediamine is an inhibitor of Na+- and K+-activated processes of Na+/K+-ATPase, i.e. the overall Na+/K+-ATPase activity, Na+-activated ATPase and K+-activated phosphatase activity, the Na+-activated phosphorylation and the Na+-free (amino-buffer associated) phosphorylation. (2) The I50 values (I50 is the concentration of inhibitor that half-maximally inhibits) increase with the concentration of the activating cations and the half-maximally activating cation concentrations (Km values) increase with the inhibitor concentration. (3) Ethylenediamine is competitive with Na+ in Na+-activated phosphorylation and with the amino-buffer (triallylamine) in Na+-free phosphorylation. Significant, though probably indirect, effects can also be noted on the affinity for Mg2+ and ATP, but these cannot account for the inhibition. (4) Inhibition parallels the dual protonated or positively charged ethylenediamine concentration (charge distance 3.7 A). (5) Direct investigation of interaction with activating cations (Na+, K+, Mg+, triallylamine) has been made via binding studies. All these cations drive ethylenediamine from the enzyme, but K+ and Mg+ with the highest efficiency and specificity. Ethylenediamine binding is ouabain-insensitive, however. (6) Ethylenediamine neither inhibits the transition to the phosphorylation enzyme conformation, nor does it affect the rate of dephosphorylation. Hence, we provisionally conclude that ethylenediamine inhibits the phosphoryl transfer between the ATP binding and phosphorylation site through occupation of cation activation sites, which are 3-4 A apart.     

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).     

Activation of Na+/Mg2+ antiport in thymocytes by cAMP.

Mg2+ efflux from Mg(2+)-loaded rat thymocytes was stimulated by 0.1 mM dibutyryl cAMP (db cAMP). The activation of Mg2+ efflux by db cAMP was more expressed at lower Mg(2+)-loading. cAMP induced only a very small increase in the concentration of intracellular free Mg2+ which cannot explain the activation of Na+/Mg2+ antiport. From these results it was concluded that cAMP increases the affinity of the Na+/Mg2+ antiporter for intracellular Mg2+, probably by phosphorylation.     

Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg(2+)-stimulated ATPase, (b) K(+)-stimulated ATPase, (c) (Na(+),K(+))-stimulated ATPase and (d) Na(+)-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0.1mum for ouabain-sensitive Mg(2+)-stimulated ATPase, 0.2mum for K(+)-stimulated ATPase, 0.1mum for (Na(+),K(+))-stimulated ATPase and 0.003mum for Na(+)-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca(2+), whereas the ouabain-sensitive Mg(2+)-stimulated ATPase activity was activated by the same concentration of Ca(2+). The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q(10) was 6.6 for K(+)-stimulated ATPase activity, 3.7 for (Na(+),K(+))-stimulated ATPase and 2.6 for Na(+)-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K(+)-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na(+) with decreasing pH. This Na(+)-independent phosphorylation at low pH was sensitive to K(+) and hydroxylamine as well as the Na(+)-dependent phosphorylation at neutral pH.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin

Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca(2+)+Mg(2+)-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2mug/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca(2+)+Mg(2+)-stimulated ATPase by increasing the Ca(2+)-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca(2+)+Mg(2+)-dependent ATPase in addition to its effect on the Ca(2+)-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca(2+)+Mg(2+)-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca(2+) concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5mum-cyclic AMP inhibited Ca(2+)+Mg(2+)-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase. Secondly, trifluoperazine (20mum) antagonized the stimulation of Ca(2+)+Mg(2+)-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase by PKI.     

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 lanthanides as competitors of Na+ and K+ in occlusion sites of renal (Na+,K+)-ATPase.

Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase. Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase. The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded. All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small. The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites. Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites. Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like. They stabilize the E1 state and compete with K+ ions. The Ki for La3+ is 0.445 microM. The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*[protein] + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids). Direct assays confirm that Tb3+ binding is nonspecific. Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+. La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition. The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+). It is 1.741 s-1 at 25 degrees C. Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments. In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.     

Role of magnesium in the (Ca2+ + Mg2+)-stimulated membrane ATPase of human red blood cells.

(Ca2+ + Mg2+)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca2+ concentration and at two different but constant Mg2+ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value for Km (for ATP) of 1-2 micron is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975. J. Membrane Biol. 22:313). Mg2+ increases both the maximal rate and the affinity for ATP, whereas Ca2+ increases the maximal rate without affecting Km for ATP. As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1 mM substrate concentration is several times faster than maximal rate of (Ca2+ + Mg2+)ATPase in red cell membranes.     

The inhibition of yeast enolase by Li+ and Na+1

The activity of yeast enolase is inhibited by Li+ and Na+. At pH 7.1, inhibition by Li+ is "mixed" with respect to Mg2+; both Vmax and Km (Mg2+) are increased by Li+. The inhibition by Li+ appears to be partial, indicating that enzyme with Li+ bound is active. The step inhibited by Li+ cannot be proton abstraction since Li+ decreases the kinetic isotope effect on Vmax. At pH 9.2, where proton abstraction is no longer partially rate-limiting, inhibiton by Li+ is competitive with respect to Mg2+. The rate of enzyme-catalyzed exchange of the C-2 hydrogen with solvent is not affected by Li+. We interpret these results as follows: Li+ (and Na+) binds to enolase and decreases the rate of at least one step in the mechanism. At pH 7.1, this step is partially rate-limiting; at pH 9.2, this step is a fast step in the reaction. The step inhibited by Li+ cannot be proton abstraction but may be release of product (phosphoenol pyruvate) or Mg2+.     

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.