A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.     

Rapid inhibition of the K(+)-sensitive phosphoenzyme of Na+/K(+)-ATPase by (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine.

Membrane-bound Na+,K(+)-ATPase (0.1 mg/ml) was incubated with the K(+)-site-directed probe (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) (Takada, J. et al. (1990) Biochim. Biophys. Acta 1037, 373-379) at 37 degrees C for 30 min in the absence of ligands, then the Na(+)-dependent phosphorylation level was examined in the presence of 10 microM [32P]ATP at 0 degrees C. The level was decreased to 50% and 0% by about 50 microM and 100 microM AU-1421, respectively. Addition of 1 mM K+ during the treatment with AU-1421 resulted in complete maintenance of the phosphorylation level. When the preincubation was performed at 0 degrees C for 10 s, even 100 microM AU-1421 did not impair the phosphorylation. In contrast to the non-phospho form of the enzyme, the K(+)-sensitive phosphoenzyme formed from ATP was immediately inhibited by the addition of AU-1421 at 0 degrees C. The reactivity of the inhibited phosphoenzyme was restored by the addition of K+. About 1 mM K+ gave the same maximal reactivity in the presence of various fixed concentrations (8-41 microM) of AU-1421, but the apparent affinity for K+ decreased simply with the increase of AU-1421 concentration. From this simple competitive relationship, the apparent Ki value of AU-1421 for the phosphoenzyme was calculated to be 7.2 microM. Compared to the non-phospho form of the enzyme, the phospho form appears to be rather susceptible to AU-1421, probably because the K(+)-site of the phosphoenzyme is exposed to the extracellular aqueous phase.     

K(+)-site-directed pyridine derivative, AU-1421, activates hydrolysis of the K(+)-sensitive phosphoenzyme of sarcoplasmic reticulum Ca(2+)-ATPase and inactivates that of K(+)-transporting ATPases.

(Z)-5-Methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, AU-1421, interacted at 0 degree C with the K(+)-sensitive phosphoenzymes of three transport ATPases, Ca(2+)-, H+/K(+)- and Na+/K(+)-ATPase. In the case of Ca(2+)-ATPase, AU-1421 at about 80 microM stimulated 6-fold the rate of splitting of the phosphoenzyme, on which K+ simply functions as an accelerator from one side of the membrane. Probably AU-1421 also simply interacts with the K(+)-binding site of the phosphoenzyme that is easily accessible from the aqueous phase. In the cases of H(+)/K(+)- and Na(+)/K(+)-ATPases, AU-1421 stabilized the phosphoenzymes which accept K+ as the translocating ion. The rate constants of dephosphorylation for H(+)/K(+)-ATPase and Na(+)/K(+)-ATPase were decreased to half by AU-1421 at about 5 and 10 microM, respectively. Presumably after binding of AU-1421 to a K(+)-recognition site of the phosphoenzyme, local motion of the peptide region near the binding site that serves to move the bound ion into the ion-transport pathway (occlusion center) might be inhibited. Thus AU-1421 may be able to distinguish two modes of K+ action on the K(+)-sensitive phosphoenzymes.     

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.     

How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Myometrial (Na+ + K+)-activated ATPase and its Ca2+ sensitivity

Ouabain-sensitive (Na+ + K+)-ATPase activity in the rat myometrial microsome fraction could only be determined following detergent treatment. The (Na+ + K+)-ATPase activity manifested by detergent treatment proved very stable even to high concentrations of NaN3, in contrast Mg+-ATPase activity was reduced to about 30 percent of the control. The major part of the Mg2+-ATPase in the myometrial membrane preparation was found to be identical with the NaN3-sensitive ATP diphosphohydrolase capable of ATP and ADP hydrolysis. This monovalent-cation-insensitive ATP hydrolysis could be extensively reduced by DMSO. Furthermore DMSO prevented the inactivation of the (Na+ + K+)-ATPase activity. 10-100 microM Ca2+ inhibited the (Na+ + K+)-ATPase activity obtained in the presence of SDS by 15-50 percent. The Ca2+ sensitivity of the enzyme was considerably decreased if the proteins solubilized by the detergent had been separated from the membrane fragments by ultracentrifugation. The inhibitory effect could be regained by combining the supernatant with the pellet. Ca2+ sensitivity of the (Na+ + K+)-ATPase activity was preserved even after removal of the solubilized proteins provided that DMSO had been applied. It appears that a factor in the plasma membrane solubilized by SDS may be responsible for the loss of Ca2+ sensitivity of the (Na+ + K+)-ATPase activity, the solubilization of which can be prevented by DMSO.     

Effects of choline on Na(+)- and K(+)-interactions with the Na+/K(+)-ATPase.

Choline chloride, 100 mM, stimulates Na+/K(+)-ATPase activity of a purified dog kidney enzyme preparation when Na+ is suboptimal (9 mM Na+ and 10 mM K+) and inhibits when K+ is suboptimal (90 mM Na+ and 1 mM K+), but has a negligible effect at optimal concentrations of both (90 mM Na+ and 10 mM K+). Stimulation occurs at low Na+ to K+ ratios, but not at those same ratios when the actual Na+ concentration is high (90 mM). Stimulation decreases or disappears when incubation pH or temperature is increased or when Li+ is substituted for K+ or Rb+. Choline+ also reduces the Km for MgATP at the low ratio of Na+ to K+ but not at the optimal ratio. In the absence of K+, however, choline+ does not stimulate at low Na+ concentrations: either in the Na(+)-ATPase reaction or in the E1 to E2P conformational transition. Together, these observations indicate that choline+ accelerates the rate-limiting step in the Na+/K(+)-ATPase reaction cycle, K(+)-deocclusion; consequently, optimal Na+ concentrations reflect Na+ accelerating that step also. Thus, the observed K0.5 for Na+ includes high-affinity activation of enzyme phosphorylation and low-affinity acceleration of K(+)-deocclusion. Inhibition of Na+/K(+)-ATPase and K(+)-nitrophenylphosphatase reactions by choline+ increases as the K(+)-concentration is decreased; the competition between choline+ and K+ may represent a similar antagonism between conformations selected by choline+ and by K+.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase.

Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.     

Dialdehyde ATP derivative as an affinity modifier of the Na+,K(+)-ATPase active site

Interaction of Na+,K(+)-ATPase from pig kidney in various conformational states with the dialdehyde analogue of ATP, alpha,alpha-(9-adenyl)-alpha'-D-(hydroxymethyl)diglycolaldehyde triphosphate ester (oATP), has been studied. This interaction leads to an enzyme modification which was shown to be of the affinity type according to the following criteria. 1. oATP can be hydrolyzed by Na+,K(+)-ATPase and prevent inhibition of ATPase activity by gamma-[4-(N-2-chloroethyl-N-methylamino)]benzylamide ATP, indicating that it interacts with Na+,K(+)-ATPase in the enzyme active site. 2. oATP irreversibly inhibits ATP-hydrolyzing activity of Na+,K(+)-ATPase; the extent of inactivation is decreased in the presence of 20 mM ATP and depends on the ion composition of the modification medium. The inhibition and ATP protection are maximal in Na+,Mg2(+)-containing buffer. 3. The value of [14C]oATP incorporation into the alpha subunit is proportional to the degree of enzyme inactivation at low (less than 0.1 mM) concentration of oATP and, on extrapolation to complete inhibition, corresponds to incorporation of 1.05 mol reagent/mol alpha subunit. 4. Tryptic hydrolysis of the isolated oATP-modified alpha subunit and subsequent separation of the peptides revealed only one labelled fragment with a molecular mass of about 10 kDa. Localization of the modified fragment in the alpha-subunit polypeptide chain is discussed. A morpholine-like structure was shown to be formed as a result of the modification.     

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase

The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl-induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state.     

Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P.     

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).     

Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein

The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP (but not AMP), and Na+, and decreased by K+, Rb+, NH+4, and LI+. Thus the fluorescence effects correlate with the ability of these groups of ligands to stabilize E1 and E2 conformations, respectively. The Na+-induced increase in fluorescence has two components: a slow, high-affinity increase of approximately 7% (K0.5 = 0.16 mM) with positive cooperativity; and a large (approximately 15%), rapid, low-affinity (K0.5 = 34 mM) increase that is not cooperative. The K0.5 for the high-affinity effect is decreased by oligomycin and increased by K+. ATP effects on the fluorescence follow Michaelis-Menten kinetics and are of high affinity (K0.5 = 0.12 microM); K+ increases the K0.5 for ATP, AMP-PNP, and ADP but does not induce cooperative behavior. K+ itself decreases the fluorescence signal by about 9%, with high affinity (K0.5 = 5 microM), showing Michaelis-menten behavior in the absence of other ligands, while with ATP, Na+, or Mg2+ present, K+ effects are cooperative and of lower affinity.     

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.     

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.     

Existence of ADP- and KCl-insensitive phosphoenzyme intermediate of Na+,K(+)-ATPase at alkaline Ph

The Na(+)-ATPase activity of Na+,K(+)-ATPase in the absence of K+ was least dependent on the sodium concentration when the pH was 9.5. Around 40% of the phosphoenzyme formed from ATP in the presence of 0.5 mM MgCl2 at alkaline pH was insensitive to both KCl and ADP. High-Na+ chase reversed this insensitivity, i.e., the phosphoenzyme became sensitive to KCl or ADP. On the other hand, phosphorylation at 0.1 mM MgCl2 instead of 0.5 mM showed at least 95% sensitivity to KCl. These observations suggest that ADP- and KCl-insensitive phosphoenzyme was formed when excess Mg++ was present during phosphorylation at alkaline pH. This phosphoenzyme might be an intermediate in the process of ATP hydrolysis.     

The effect of cytoplasmic K+ on the activity of the Na+/K(+)-ATPase.

Experiments with the reconstituted (Na+ + K+)-ATPase show that besides the ATP-dependent cytoplasmic Na(+)-K+ competition for Na+ activation there is a high affinity inhibitory effect of cytoplasmic K+. In contrast to the high affinity K+ inhibition seen with the unsided preparation at a low ATP especially at a low temperature, the high affinity inhibition by cytoplasmic K+ does not disappear when the ATP concentration an-or the temperature is increased. The high affinity inhibition by cytoplasmic K+ is also observed with Cs+, Li+ or K+ as the extracellular cation, but the fractional inhibition is much less pronounced than with Na+ as the extracellular cation. The results suggest that either there are two populations of enzyme, one with the normal ATP dependent cytoplasmic Na(+)-K+ competition, and another which due to the preparative procedure has lost this ATP sensitivity. Or that the normal enzyme has two pathways for the transition from E2-P to E1ATP. One on which the enzyme with the translocated ion binds cytoplasmic K+ with a high affinity but not ATP, and another on which ATP is bound but not K+. A kinetic model which can accommodate this is suggested.