Studies on conformational changes in Na,K-ATPase labeled with 5-iodoacetamidofluorescein

The rates of individual steps in the reaction cycle of dog kidney Na,K-ATPase labeled with iodoacetamidofluorescein (IAF) were measured using the fluorescence stopped-flow technique. The maximal rate of the fluorescence quenching accompanying ATP hydrolysis at 20 degrees C in the presence of K+ is 66.3 s-1, while the turnover rate in the same conditions is 15.5 s-1. The rate without K+ is slightly lower. Unexpectedly, at very high ionic strength, K+ accelerates the rate 2-fold. The fluorescence change appears to be associated with the E1P----E2P transition. The results are consistent with the classical Albers-Post scheme but do not support recent criticisms that E1P is kinetically incompetent in the presence of Na+ plus K+. As expected, in the absence of ATP the rate of E2(K)----E1Na was very slow (0.2 s-1) but was greatly accelerated by ATP (maximal rate 15.9 s-1) with low affinity (K0.5 = 196 microM). It was concluded that E2(K)----E1 is the slowest step of the cycle, even at nonlimiting ATP concentrations. The rate of E1K----E2(K) for both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme was stimulated by K+ acting with low affinity, but not at all by ATP at 5 microM. Whereas the maximal rate with IAF-enzyme (271 s-1) was similar to previous work, the K+ affinity was significantly higher. Fluorescence signals accompanying hydrolysis of acetyl phosphate with both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme have similar rates, 5.25 s-1 and 4.06 s-1, respectively. A species difference was observed between dog and pig kidney Na,K-ATPase in that both enzymes are labeled with IAF but only in dog enzyme were conformational transitions associated with fluorescence changes. Therefore, the IAF-labeled dog kidney enzyme is the preparation of choice for measuring fluorescence changes accompanying ATP hydrolysis.     

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.     

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).     

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.     

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.     

Defective conformational response in a selectively trypsinized (Na+ + K+)-ATPase studied with tryptophan fluorescence

1. Monitoring protein conformations of purified (Na+ + K+)-ATPase with intrinsic fluorescence we have examined if altered conformational responses accompany the defective catalytic and transport processes in selectively modified 'invalid' (Na+ + K+)-ATPase which is obtained by graded tryptic digestion of the Na+ form of the protein. 2. The protein fluorescence intensity of the K+ form (E2K) of both control and invalid (Na+ + K+)-ATPase is 2--3% higher than that of the Na+ form (E1Na). By varying the NaCl concentration we found evidence for different fluorescence intensities of the two phosphoenzymes; E2P has the same fluorescence intensity as E2K and the intensity of E1P is similar to that of E1Na. The fraction of phosphoenzyme present as E2P can therefore be determined as the amplitude of the fluorescence change accompanying phosphorylation in the absence of K+ divided by the amplitude of the full response to K+. 3. Titration of the fluorescence responses of the invalid (Na+ + K+)-ATPase shows that the tryptic split alters the noise of the equilibria between the cation-bound conformations, E1Na and E2K, and between the phosphoforms, E1P and E2P, in the direction of the E1 forms. 4. Vanadate binds to the Mg2+-bound form of E2K and prevents further changes in fluorescence intensity of the protein. The conformative responses of invalid (Na+ + K+)-ATPase are insensitive to vanadate in agreement with the reduced vanadate binding affinity of this enzyme. 5. The defective conformative response of the invalid (Na+ + K+)-ATPase in relation to its catalytic defects, reduced Na+ transport, and insensitivity to vanadate suggest that the transitions between Na+ forms (E1) and K+ forms (E2) of the protein are coupled to the catalytic and transport reactions of the (Na+ + K+)-pump.     

Binding of monovalent cations induces large changes in the secondary structure of Na+,K+-ATPase as probed by Raman spectroscopy

Raman spectra of active Na+,K+-ATPase from pig kidney in media containing Na+ (E1), K+ (E2) or without exogenous ions (E1 conformation) were recorded in order to calculate the changes in the enzyme's secondary structure induced by binding of monovalent cations. It is demonstrated that: (i) K+ binding to the E1 form of the enzyme leads to conversion of approximately 100 peptide groups from the beta-structure to alpha-helical conformation; (ii) the transition is reversible and fully reproducible in the E1----E2----E1 and E2----E1----E2 experimental schemes. Predictional calculations revealed polypeptide chain segments involved in the alpha----beta transformations. These segments reside mainly in the two highly conserved regions of the alpha-subunit in the cytoplasmic domain of Na+,K+-ATPase. A possible role for the beta-subunit is discussed.     

Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change

The (Na+ + K+)ATPase is inhibited by the bee venom polypeptide, melittin. KCl and NaCl protect the enzyme from melittin inhibition. Analysis of the K+ and Na+ protection against melittin inhibition suggested a kinetic model which was consistent with slowly reversible melittin binding, and mutually exclusive binding of melittin with K+ and Na+. Accordingly, in the absence of salt, the KI for melittin inhibition = 1.2 microM, and the protection by KCl occurs with a KA,KCl = 0.6 mM. The protection by NaCl occurs with a KA,NaCl = 15 mM. Melittin inhibition of enzyme activity is due to direct interactions with the (Na+ + K+)ATPase, as demonstrated by photolabeling with [125I]azidosalicylyl melittin, which labeled the alpha subunit, but not the beta subunit of the (Na+ + K+)ATPase. Melittin and KCl reduced the extent of labeling. In non-covalent binding studies using [125I]azidosalicylyl melittin, the stoichiometry of binding was 1.6 melittin per (Na+ + K+)ATPase. Ligand-induced conformational changes of FITC-labeled (Na+ + K+)ATPase were examined in the presence and absence of melittin. K+ alone or melittin alone caused a fluorescence intensity quenching consistent with formation of an E2 form of the enzyme. The NaCl-induced (E2----E1) fluorescence intensity changes were maximal when the enzyme was treated with K+. NaCl-induced fluorescence changes did not occur when the enzyme was treated with melittin in the absence of K+. However, when K+ was present before the addition of melittin, NaCl-induced fluorescence intensity increases were observed, which were dependent upon the concentration of K+ in the preincubation mixture. The results of the labeling and conformational studies support the kinetic model and suggest a mechanism for inhibition of ion pumps by (poly)peptides.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

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.     

5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover

The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively.     

Uncoupling the red cell sodium pump by proteolysis

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Free protons do not substitute for sodium ions in buffer-mediated phosphorylation of (Na+ + K+)-ATPase

In view of our recent finding of imidazole-activation of the phosphorylation of (Na+ + K+)-ATPase and the suggestion by others of an activating role of protons, in lieu of sodium ions, in the overall hydrolytic and phosphorylation processes of the enzyme, we have investigated the effect of pH on the phosphorylation process. No indication of proton activation is found. Rather, phosphorylation at low pH in the absence of Na+ is dependent on the buffer concentration. Imidazole-H+ stimulated phosphorylation at pH 5 reaches the same maximal steady-state level as Na+-stimulated phosphorylation. Low pH also elicits Tris-H+ stimulated phosphorylation, but due to a simultaneous inhibitory effect of this buffer the maximal steady-state level is no more than 50% of the Na+-stimulated phosphorylation level. Protons inhibit rather than activate phosphorylation. Upon decreasing the pH from 7 to 5, we observe for all ligands, whether activating or inhibiting phosphorylation (ATP, Na+, protonated imidazole, Mg2+ and K+), a decrease in affinity (largest for Mg2+) and a decrease in the maximal steady-state phosphorylation capacity. The effects of Na+ and imidazole-H+ on the phosphorylation step have been compared with those on the E2----E1 conformational change, which leads to the phosphorylation step. The different pH-dependence of the affinities for Na+ and protonated buffer in the E2----E1 transition suggests that there are separate activation sites with different pK values for Na+ and the buffer cation. The above findings rule out a role of free protons as a substitution for Na+ in the phosphorylation process.     

Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.     

Phosphoenzyme decomposition in dog cardiac sarcoplasmic reticulum Ca2+-ATPase

A five-syringe quench-flow apparatus was used in the transient-state kinetic study of intermediary phosphoenzyme (EP) decomposition in a Triton X-100 purified dog cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase at 20 degrees C. Phosphorylation of the enzyme by ATP in the presence of 100 mM K+ for 116 ms gave 32% ADP-sensitive E1P, 52% ADP- and K+-reactive E2P, and 16% unreactive residual EPr. The EP underwent a monomeric, sequential E1P 17 s-1----E2P 10.5 s-1----E2 + Pi transformation and decomposition in the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid quenched Ca2+-devoid medium. The calculated rate constant for the total EP (i.e., E1P + E2P) dephosphorylation was 7.8 s-1. The E1P had an affinity for ADP with an apparent Kd congruent to 100 microM. When the EP was formed in the absence of K+ for 116 ms, no appreciable amount of the ADP-sensitive E1P was detected. The EP comprised about 80% ADP- and K+-reactive E2P and 20% residual EPr, suggesting a rapid E1P----E2P transformation. Both the E2P's formed in the presence and absence of K+ decomposed with a rate constant of about 19.5 s-1 in the presence of 80 mM K+ and 2 mM ADP, showing an ADP enhancement of the E2P decomposition. The results demonstrate mechanistic differences in monomeric EP transformation and decomposition between the Triton X-100 purified cardiac SR Ca2+-ATPase and deoxycholate-purified skeletal enzyme [Wang, T. (1986) J. Biol. Chem. 261, 6307-6319].     

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

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

Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.     

The E1----E2 transition of Ca2+-transporting ATPase in sarcoplasmic reticulum occurs without major changes in secondary structure. A circular-dichroism study.

C.d. spectroscopy was used to investigate the structures of Ca2+-ATPase (Ca2+-transporting ATPase) in the E1 and E2 states in native, in fluorescein isothiocyanate (FITC)-labelled and in solubilized sarcoplasmic reticulum (SR) preparations. The E1 state was stabilized by 100 microM-Ca2+ and the E2 state by 0.5 mM-Na3 VO4 and 0.1 mM-EGTA. There were no significant differences detected in the c.d. spectra and the calculated secondary structures between the E1 and E2 states in any of the three types of preparations. The FITC-labelled SR did show the characteristic changes in FITC fluorescence on addition of Ca2+ or vanadate, indicating that the preparation was competent for E1----E2 transitions. Therefore the absence of changes in the c.d. spectra implies that the E1----E2 transition in the Ca2+-ATPase does not involve a major net rearrangement of the polypeptide backbone conformation.