E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase.

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.

E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate.

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.

Interpretation of extraordinary kinetics of Na(+)-K(+)-ATPase by a phase change.

We interpret at a molecular level an extraordinary response in the transient kinetics of the phosphointermediate of Na(+)-K(+)-ATPase (I. Klodos, R. L. Post, and B. Forbush III. J. Biol. Chem. 269: 1734-1743, 1994). The phosphointermediate comprises two principal states. The partition between these states varies with salt concentration. A jump in salt concentration changes the partition of some of the molecules more rapidly than they interconvert in a steady state at constant salt concentration. We propose that interconversion is limited by free volume in the lipid of the surrounding membrane. This lipid is partitioned into phases that vary with salt concentration. Free volume is larger at the interface between these phases than within the phases themselves. Na(+)-K(+)-ATPase molecules are distributed at random in the membrane. When the phase boundary moves in response to a jump in salt concentration, it crosses some Na+ -K+ -ATPase molecules, which transiently experience an increase in free volume of the surrounding lipid. Thus their phosphointermediate states equilibrate more rapidly than at a constant salt concentration. Functional and structural heterogeneity of Na(+)-K(+)-ATPase molecules is discussed.

Fluorescent styryl dyes as probes for Na,K-ATPase reaction mechanism: significance of the charge of the hydrophilic moiety of RH dyes.

The fluorescence responses of a series of potential-sensitive styryl-based dyes (either zwitterionic RH160, RH421, di-4-ANEPPS, or positively charged RH795, RH414, RH461) to phosphorylation of Na,K-ATPase from ATP or inorganic phosphate, and ouabain binding to phospho- or dephosphoforms, have been characterized and compared in broken membrane preparations of the enzyme. Zwitterionic dyes were more sensitive to molecular events in the Na,K-ATPase reaction cycle than positively charged dyes, but the net charge did not affect the sensitivity of the dyes to a transmembrane electric field. The major part of the response of the zwitterionic dyes to formation of phosphoenzymes was due to a change in the quantum yield of fluorescence. Computer modeling of dyes with identical chromophore structure, and experimental characterization of their optical properties in bulk solvents, revealed two general trends: (1) the absorption maximum of the zwitterionic dye was blue-shifted with respect to the positively charged dye; (2) the quantum yield of the zwitterionic dye was higher and the fluorescence lifetime was longer than that for the positively charged dye. Spectral properties of the dyes in the membrane depended on the presence of Na,K-ATPase. We suggest, that (1) electrostatic interactions between the enzyme and the hydrophilic headgroup of the dye by changing the charge of hydrophilic moiety and thus modifying the net charge of the dye molecule cause both the spectral shifts and the changes in the quantum yield, and (2) interactions between the styryl dyes and the Na,K-ATPase depend on the conformational state of the enzyme.

Influence of intramembrane electric charge on Na,K-ATPase.

Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland. Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions. Phosphoenzyme interactions with extracellular cations were evaluated through K(+)-, ADP-, and Na(+)-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na(+)-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB- had opposite effects. Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-D-glucamine or Tris+, but were unaffected by the replacement of Cl- by other anions. Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field.

Kinetic heterogeneity of phosphoenzyme of Na,K-ATPase modeled by unmixed lipid phases. Competence of the phosphointermediate.

Interconversion of phosphoenzyme resistant to K+ and sensitive to ADP (E1P) and phosphoenzyme resistant to ADP and sensitive to K+ (E2P) was studied in bovine brain and dog and pig kidney. The kinetics of dephosphorylation were observed by chasing phosphoenzyme formed from [32P]ATP with unlabeled ATP with or without ADP or K+. Phosphorylation in 0.6-1.0 M NaCl produced mostly ADP-sensitive potassium-insensitive E1P. A potassium chase of this phosphoenzyme exposed its rate of conversion to potassium-sensitive ADP-insensitive E2P. At 20 degrees C the rate constant was approximately 1 s-1. Simultaneous dilution of [NaCl] in the chase to 100 mM increased the constant to approximately 60 s-1, which probably qualifies E1P as an intermediate in Na,K-ATPase activity. Anions inhibited conversion according to a Hofmeister series. Na+ had no specific effect. At 0 degrees C the rate constant was < 0.4 s-1, but downward jumps in [salt] produced an acceleration to > 1 s-1 for < 3 s followed by a return to the slow rate. The rapid rate would qualify E1P to participate in Na,K-ATPase activity if this rapid state participates in the normal reaction cycle. Phosphorylation in 0.02-0.1 M NaCl produced mostly E2P. Upward jumps in [NaCl] converted E2P to E1P equally rapidly and transiently. Oligomycin and high [salt] cooperated in stabilizing E1P. Jumps in [salt] greatly and transiently increased the rate of conversion of one form of the phosphoenzyme to the other. This extraordinary result required heterogeneous kinetics. A model is proposed based on control of enzyme conformation by changes in separate unmixed phases of the lipid of the membrane.

The influence of membrane charge on the kinetic properties of Na,K-ATPase.

Lipophilic ions modify the affinity of the cation binding sites of the membrane-bound Na,K-ATPase. We studied the effect of the lipophilic ions tetraphenyl-phosphonium (TPP+) and tetraphenylboron (TPB-) on the binding of Na+ and K+ to the cation site(s) that are exhibited by the enzyme during the catalytic cycle: the high-affinity (inside) Na-binding site, site I, the low-affinity (outside) Na-leaving site, site II, and the high-affinity (outside) K-site, site III. Site I: In the presence of TPP+ (positive charge added to the lipid environment) a higher Na(+)-concentration was needed to obtain phosphorylation of the enzyme, whereas in the presence of TPB- (negative charge added to the lipid environment) phosphorylation was obtained at a lower Na(+)-concentration, but the change in apparent K0.5 for Na+ was small, (K0.5Na,TPP = 0.180 mM and K0.5Na,TPB = 0.07 mM), indicating only a minor influence of membrane charge on Na+ binding to site I. Site II: Compared to control conditions, more Na+ was required to inhibit ATP-hydrolysis and to increase the steady-state level of ADP-sensitive phosphoenzyme when TPP+ was present, and the opposite was observed with TPB-, indicating a strong influence of membrane charge on the Na+ occupancy of site II. Site III: TPP+ induced a significant decrease both in the rate of K-dependent dephosphorylation of preformed E32P and in the K+ affinity. The effect of TPP+ on the ATP hydrolysis rate strongly resembled the effect of decreasing [KCl]. The results indicated a pronounced effect of adding positive charge to the lipid environment of site III.(ABSTRACT TRUNCATED AT 250 WORDS)

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant.

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.

Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates.

(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM. (2) In the absence of ADP the dephosphorylation curve was linear in a semilogarithmic plot almost from t = 0, whereas by addition of ADP a biphasic behaviour was obtained. The slope of the slow phase of dephosphorylation was virtually independent of the ADP concentration. (3) The results were analysed by the mathematical equation corresponding to the simplest possible model for the interconversion and breakdown of the phosphointermediates: (formula: see text) where alpha, beta, H and G are functions of all the rate constants and H and G furthermore are functions of the initial values for [E1P] and [E2P]. (4) The analysis confirmed the model and enabled the determination of all the rate constants. (5) k-1 was found to be equal to k'-1 + k"-1 . [ADP] indicating an ADP-independent 'spontaneous' dephosphorylation of E1P. The rate constant for this process was close to that for dephosphorylation of E2P, i.e., k'-1 congruent to k3. Also the value of k"-1 was determined. (6) k3 was found to be at least 10 . k-2. The implication of this for the role of the E1P to E2P transition in the Na+ + K+)-stimulated ATP hydrolysis will be discussed in detail in the following paper (Plesner, I.W., Plesner, L., Nørby, J.G. and Klodos, I. (1981) Biochim. Biophys. Acta 643, 483--494). (7) A refinement of the model, accounting for the effect of Na+ on the steady-state ratio between [E1P] and [E2P] is proposed: (formula: see text). At [Na+] = 150 mM as used here, E1P(Na) and E'1P are assumed to be in rapid equilibrium. (8) Comparison of our results with those of others underlines the general validity of the conclusions of the present paper.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model.

A steady-state kinetic investigation of the effect of K+ on the Na+-enzyme activity of the (Na+ + K+)-ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1. K+ inhibits the Na+-enzyme (the enzyme activity measured at micromolar substrate concentrations in the presence of Na+). The inhibition of non-competitive at low and competitive at higher K+ concentrations and is enhanced by free Mg2+. 2. The results indicate that the Na+-enzyme at steady-state tends to be accumulated in an enzyme-potassium complex when K+ is added. 3. The enzyme-potassium complex, in turn, binds Mg2+ in a dead-end fashion. The dissociation constant for the enzyme-K-Mg complex, estimated from the data, is 7.2 mM. The same value was obtained earlier for the Mg2+ inhibition constant of the substrate-free form of the (Na+ + K+)-enzyme (the enzyme activity measured with Na+ and K+ and at millimolar substrate concentrations) suggesting that the two constants describe the same equilibrium. 4. On the basis of the known (optimal) activity of the (Na+ + K+)-ATPase, relative to that of the Na+-ATPase, a rate constant condition is found which must be met if the Post-Albers kinetic scheme is to satisfy the data. Kinetic data for the phosphoenzyme indicate that this condition is not satisfied. 5. On the basis of the kinetic results a model for the hydrolytic action of (Na+ + K+)-ATPase is proposed. This model encompasses the Post-Albers scheme but contains two distinctive hydrolysis cycles (an 'Na+-enzyme cycle' and a '(Na+ + K+)-enzyme cycle') with widely different affinities for the substrates. Only one of the cycles (the Na+-enzyme cycle) involves acid-stable phosphorylated enzyme intermediates at discernible steady-state concentrations. Which of the two main cycles is predominant in any particular system is determined by the concentration of ligands and substrates. 6. According to this scheme, an enzyme preparation may exhibit both a high (Na+-enzyme) and a low ((Na+ + K+)-enzyme) substrate affinity, without the necessity of assigning more than one substrate site to a particular enzyme unit at any one time.