The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.     

Isotope-dilution analysis of rate-limiting steps and pools affecting the incorporation of thymidine and deoxycytidine into cultured thymus cells

1. Ox brain microsomal fractions were labelled with [(32)P]ATP in the presence of Na(+) and the reaction was stopped with sodium dodecyl sulphate. The Na(+)-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [(32)P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na(+)-dependent label emerged in a peak corresponding to protein of molecular weight 570000-580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na(+)-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [(32)P]ATP in the absence of Na(+) revealed three labelled peaks, one of which corresponded in position to the Na(+)-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na(+)-dependent label. 4. Di-isopropyl [(32)P]phosphorofluoridate also labelled the band corresponding to the Na(+)-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50-60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.     

Phosphate binding by cerebral microsomes in relation to adenosine-triphosphatase activity

1. Microsomes prepared from guinea-pig and ox brain were incubated for periods of a few seconds with low concentrations of Mg-[(32)P]ATP, the reaction was stopped with trichloroacetic acid and determinations were made of the phosphate bound to the acid-washed, and in some cases solvent-extracted, residue. 2. At 20 mum-ATP, at 37 degrees and in the presence of Na(+) ions, 30-50 mumumoles of phosphate/mg. of microsomal protein were bound by the preparation within 1 sec. of starting the reaction; little further change in level occurred until hydrolysis of ATP exceeded 50%, when the bound phosphate began to decline fairly rapidly to the zero-time value. 3. At 20mum-ATP without Na(+) ions present or in the presence of K(+) ions, the level of bound phosphate increased gradually and did not decline as ATP hydrolysis approached completion. 4. Potassium ions either inhibited the formation of Na(+)-dependent bound phosphate or, when added during the course of the reaction, rapidly reduced its level. 5. At 200 mum-ATP the bound phosphate formed in the presence of Na(+) ions appeared to consist of a mixture of the unstable Na(+)-dependent type and the stable type requiring only Mg(2+) ions for its formation. 6. Non-radioactive ATP added during the course of the reaction at 20 mum-ATP with Na(+)ions present rapidly discharged virtually all the bound (32)P counts; at 200 mum-ATP only a proportion of the label was similarly discharged. The Na(+)-dependent bound phosphate is therefore turning over, in contrast with that formed in the absence of Na(+)ions, which proved more stable. 7. The Na(+)-dependent bound phosphate was not in the form of ATP; experiments with [(14)C]ATP instead of [(32)P]ATP showed a small and invariable binding of ATP by the preparation unaffected by Na(+) ions or time of incubation. 8. Under the usual conditions employed in this work ouabain stimulated formation of Na(+)-dependent bound phosphate when Na(+) ions were suboptimum and inhibited it when optimum Na(+) ions were present. 9. The Na(+)-dependent binding reaction under present conditions did not involve incorporation into phosphorylserine groups. 10. The relation of the findings to the (Na(+),K(+))-ATPase of the preparation, and to observations in brain slices appearing to implicate phosphorylserine groups in cation transport, is discussed.     

Enzyme transfer of phosphate from adenosine triphosphate to protein-bound serine residues in cerebral microsomes

1. Microsomes from guinea-pig brain grey matter were incubated with [(32)P]ATP at 3mm concentration and the phosphate bound to the acid-washed, lipid-free residue was determined. 2. The binding process was Mg(2+)-dependent and resulted in the transfer of about 1-2 mmumoles of phosphate/mg. of protein/min. Under the conditions used univalent cations (Na(+),K(+) and Li(+)) inhibited the binding. 3. An unspecified proportion of this bound phosphate could be recovered in protein-derived phosphorylserine. The yield of labelled phosphorylserine was also decreased by univalent cations. 4. The bound phosphate formed with 3mm-MgATP was stable; addition of Na(+) or K(+) ions to the already labelled preparation had no effect on the bound phosphate level. 5. Bound phosphate was also formed when a solubilized fraction of the microsomes was incubated with ATP; univalent cations also inhibited this process. 6. p-Chloromercuribenzoate reduced the binding by about 25%; the inhibition was restored by cysteine.     

The partial purification of sodium-plus-potassium ion-dependent adenosine triphosphatase from the gills of Anguilla anguilla and its inhibition by orthovanadate.

1. (Na+ +K+)-dependent ATPase was partially purified from eel gills by a procedure in which the microsomal fraction of crude preparations of chloride cells was selectively extracted with sodium dodecyl sulphate. 2. The microsomal specific activity was increased 2-fold during optimal treatment with detergent. 3. The final preparation (56% pure) had a specific activity of 341 mumol of ATP hydrolysed/h per mg of protein and a turnover number of 3560 min-1. The number of ouabain-binding sties equalled the number of sites phosphorylated by ATP. 4. Both sodium orthovanadate and ouabain inhibited the purified preparation more than the microsomal fraction, vanadate being more effective on an equimolar basis than ouabain. 5. Inhibition by orthovanadate was not enhanced at 28 mM-as compared with 1mM-MgCl2 and was not reversed by beta-adrenergic agonists (cf. Josephson & Cantley (1977) Biochemistry 16, 4572--4578). 6. Of various other metallic oxyanions tested only niobate proved an effective inhibitor of the enzyme although this anion was less effective than orthovanadate. 7. Orthovanadate partially inhibited phosphorylation of the enzyme by ATP in the presence of 28 mM-MgCl2.     

Molecular mechanism of the ATP synthase's F(o) motor probed by mutational analyses of subunit a

The most prominent residue of subunit a of the F(1)F(o) ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum), which was reported to permit no substitution with retention of ATP synthesis or H(+)-coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P.modestum a subunit catalyse ATP-driven Na(+) transport above or below pH 8.0, respectively. Reconstituted F(o) with either mutation catalysed 22Na(+)(out)/Na(+)(in) exchange with similar pH profiles as found in ATP-driven Na(+) transport. ATP synthase with an aR227A substitution catalysed Na(+)-dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na(+) transport. This suggests that in the mutant the dissociation of Na(+) becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na(+) concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na(+) from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, F(o) with the aR227A mutation did not catalyse 22Na(+)(out)/Na(+)(in) exchange at high internal sodium concentrations, and (ii) at a constant (Delta)pNa(+), 22Na(+) uptake was inhibited at elevated internal Na(+) concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na(+) even into compartments of high Na(+) concentration.     

Low affinity superphosphorylation of the Na,K-ATPase by ATP.

Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

Halenaquinol, a natural cardioactive pentacyclic hydroquinone, interacts with sulfhydryls on rat brain Na(+),K(+)-ATPase

Halenaquinol inhibited the partial reactions of ATP hydrolysis by rat brain cortex Na(+),K(+)-ATPase, such as [3H]ATP binding to the enzyme, Na(+)-dependent front-door phosphorylation from [gamma-(33)P]ATP, and also Na(+)- and K(+)-dependent E(1)<-->E(2) conformational transitions of the enzyme. Halenaquinol abolished the positive cooperativity between the Na(+)- and K(+)-binding sites on the enzyme. ATP and sulfhydryl-containing reagents (cysteine and dithiothreitol) protected the Na(+),K(+)-ATPase against inhibition. Halenaquinol can react with additional vital groups in the enzyme after blockage of certain sulfhydryl groups with 5,5'-dithio-bis-nitrobenzoic acid. Halenaquinol inhibited [3H]ouabain binding to Na(+),K(+)-ATPase under phosphorylating and non-phosphorylating conditions. Binding of fluorescein 5'-isothiocyanate to Na(+),K(+)-ATPase and intensity of fluorescence of enzyme tryptophanyl residues were decreased by halenaquinol. We suggest that interaction of halenaquinol with the essential sulfhydryls in/or near the ATP-binding site of Na(+),K(+)-ATPase resulted in a change of protein conformation and subsequent alteration of overall and partial enzymatic reactions.     

Further observations on the inhibitory effect of extracellular potassium ions on glycine uptake by mouse ascites-tumour cells

1. The initial rate of uptake of glycine by the tumour cells was measured as a function of the Na(+) and K(+) concentrations in the solution in which the cells were suspended. When [Gly] was 1mm or 12mm, the rate in the absence of Na(+) was independent of [K(+)] and about 3% or 10% respectively of the rate when [Na(+)] was 150m-equiv./l. 2. The Na(+)-dependent glycine entry rate, v, at a given value of [Na(+)] was successively lowered when [K(+)] was increased from 8 to 47 to 96m-equiv./l. A kinetic analysis indicated that K(+) competitively inhibited the action of Na(+). The results were in fair agreement with previous determinations of the kinetic parameters. 3. The presence of 2mm-sodium cyanide and 10mm-2-deoxyglucose lowered the cellular ATP content to less than 3% of the value in the respiring cells. Although v was then about 50% smaller, the relative effects of K(+) and Na(+) on the system were similar to those observed during respiration. 4. A theoretical analysis indicated that the variation of v with [K(+)] is not a reliable guide to the extent to which the K(+) gradient between the cells and their environment may contribute to the net transport of glycine.     

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.     

Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate

When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.     

Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin.

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.     

Effect of intravesicular monovalent cations on the steady state of the phosphoenzyme of adenosine triphosphatase dependent on sodium and potassium ions in inside-out plasma-membrane vesicles.

Phosphorylation of the ATPase dependent on Na+ and K+ is promoted through the synergistic action of cations on both sides of the membrane. This phenomenon has been observed in plasma membrane vesicles isolated from sheep-kidney outer medulla which accept ATP from the outside surface (inside-out) and which are tight for sodium ions. In these inside-out vesicles phosphorylating capacity is low even in the presence of 100 mM extravesicular sodium chloride as is turnover of the enzyme. The level of the phosphoenzyme and the transient release of inorganic phosphate from the phosphoenzyme increases several-fold when sodium chloride is allowed to equilibrate over the membrane, 25 mM intravesicular NaCl is necessary to obtain the half-maximum level of the phosphoenzyme. This result shows that intravesicular (= extracellular) low affinity sites are involved in the phosphorylation. Intravesicular potassium ions modify the activating action of Na+ on the phosphorylation by increasing the steady state of the phosphoenzyme at low intravesicular sodium ion concentrations. This suggests that Na+ and K+ compete with each other for the intravesicular cation-binding site.     

A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na(+)-dependent phosphate absorption

2'-Phosphophloretin (2'-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2'-PP, on Na(+)-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2'-PP decreased Na(+)-dependent phosphate uptake into rabbit BBMV with an IC(50) of 55 nM and into rat BBMV with an IC(50) of 58 nM. 2'-PP did not affect Na(+)-dependent glucose, Na(+)-dependent sulfate, or Na(+)-dependent alanine uptake by rabbit intestinal BBMVs. 2'-PP inhibition of rabbit intestinal BBMV Na(+)-dependent phosphate uptake was sensitive to external phosphate concentration, suggesting that 2'-PP inhibition of Na(+)-dependent phosphate uptake was competitive with respect to phosphate. Binding of [(3)H]2'-PP to rabbit intestinal BBMV was examined. Binding of [(3)H]2'-PP was Na(+)-dependent with a K(0.5) for Na(+)(Na(+) concentration for 50% 2'-PP binding) of 30 mM. The apparent K(s) for Na(+)-dependent [(3)H]2'-PP binding to rabbit BBMVs was 58 nM in agreement with the IC(50) for 2'-PP inhibition of Na(+)-dependent phosphate uptake. These results indicate that 2'-PP bound to rabbit or rat intestinal BBMV Na(+)-phosphate cotransporter and inhibited Na(+)-dependent phosphate uptake. In rats treated with 2'-PP by daily gavage, the effect of 2'-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2'-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2'-PP did not alter serum calcium or serum glucose. The apparent IC(50) for 2'-PP in vivo was 3 microM.     

Tightness and orientation of vesicles from guinea-pig kidney estimated from reactions of adenosine triphosphatase dependent on sodium and potassium ions.

In order to study the "sidedness" of the ligands of the Na+, K+-ATPase in the phosphorylation from [32P]ATP, tight vesicles were prepared from guinea pig kidney and partially purified by a two-stage sucrose and Ficoll gradient centrifugation procedure. These vesicles were derived presumably from plasma membrane fragments resealed after the initial disruption of the cells during homogenization. Tightness of the vesicles was estimated according to activation by the nonionic detergent, Triton X-100. Treatment with Triton X-100 increased both the activity of the Na+, K+-ATPase and its Na+-dependent phosphorylation from [32P]ATP at least three-fold. Activation of both functions also appeared when the vesicles were shocked osmotically. These results suggest that the preparation contains a major population of tight normal vesicles (approximately 75%) in which the phosphorylation site faces the intravesicular solution. In the response to ouabain breakdown of the phosphoenzyme was inhibited in vesicles treated with Triton X-100 but not in intact ones as if ouabain could not get to its binding site. Correspondingly in phosphorylation from ATP pretreatment with ouabain in the presence of inorganic phosphate produced less inhibition in intact vesicles than in those disrupted with Triton X-100 beforehand. These data suggest the presence of an everted vesicle fraction in the preparation (approximately 20%). Apparently only a small fraction of the vesicles was leaky. In the everted vesicles the action of K+ on the phosphoenzyme was slow. In order to accelerate the dephosphorylation in intact vesicles as effectively as in disrupted ones, K+ had to be added before the start of phosphorylation. This supports the view that K+ was acting from the side of the membrane opposite to that where the gamma-phosphoryl group was accepted from ATP.     

Potassium ion-stimulated and sodium ion-dependent adenosine diphosphate–adenosine triphosphate exchange activity in a kidney microsomal fraction

1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed.     

Reaction of (Na+ + K+)-dependent adenosine triphosphatase with inorganic phosphate. Regulation by Na+, K+, and nucleotides

Effects of Na+, K+, and nucleotides on Mg2+-dependent phosphorylation of (Na+ + K+)-dependent adenosine triphosphatase by Pi were studied under equilibrium conditions. Na+ was a linear competitive inhibitor with respect to Mg2+ and a mixed inhibitor with respect to Pi. K+ was a partial inhibitor; it interacted with positive cooperativity and induced negative cooperativities in the interactions of Mg2+ and Pi with the enzyme. Adenyl-5'-yl (beta, gamma-methylene)diphosphonate, a nonhydrolyzable analog of ATP, interacted with negative cooperativity to inhibit phosphorylation in competition with Pi. ATP was also a competitive inhibitor. Na+ and K+ acted antagonistically, Na+ and nucleotides inhibited synergistically, and K+ and nucleotides were mutually exclusive. In the presence of ouabain, when nucleotides were excluded from the site inhibiting phosphorylation, a low affinity regulatory site for nucleotides became apparent, the occupation of which reduced the rate of dephosphorylation and the initial rate of phosphorylation of the enzyme without affecting the equilibrium constant of the reaction of Pi with the ouabain-complexed enzyme. The regulatory site was also detected in the absence of ouabain. The data suggest that catalytic and transport functions of the oligomeric enzyme may be regulated by homotropic and heterotropic site-site interactions, ligand-induced slow isomerizations, and distinct catalytic and regulatory sites for ATP.     

Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments

The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.     

K(+)- and Mg2(+)-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase.