The relipidation of delipidated Na,K-ATPase. An analysis of complex formation with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine.

Enzymatically inactive, delipidated Na,K-ATPase from dogfish rectal glands was titrated with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine. The process of relipidation has the following characteristic properties. Enzymatic activities reappear independently of each other: first the phosphatase, then the ATPase. The properties of the phosphatase regenerated depend on the ratio of lipid/protein used; the ATPase seems to be independent of this ratio. The simplest model that is consistent with the above results and with the shapes of the titration curves, has the following requirements. Firstly, the enzyme is composed of two subunits that, as far as lipid binding is concerned, are identical and independent of each other. Secondly, lipid adds onto the enzyme as preformed clumps of 25 molecules of phosphatidylcholine or 18 molecules of phosphatidylethanolamine. Thirdly, each subunit binds two clumps of lipid, and binding shows positive cooperativity. Fourthly, when either subunit becomes saturated with lipid, the enzyme exhibits one form of phosphatase. Fifthly, when both subunits are saturated with lipid, the enzyme exhibits a second form of phosphatase and ATPase. The data and their analysis according to this model lead to the suggestion that Na,K-ATPase is a functional dimer, the interaction between subunits being influenced by the Na+ and K+ concentrations in the medium: K+ favouring the functional independence of the subunits and Na+ favouring their functional interaction.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

Structure-function relations of interactions between Na,K-ATPase,the gamma subunit,and corticosteroid hormone-induced factor

Corticosteroid hormone-induced factor (CHIF) and the gamma subunit of the Na,K-ATPase (gamma) are two members of the FXYD family whose function has been elucidated recently. CHIF and gamma interact with the Na+ pump and alter its kinetic properties, in different ways, which appear to serve their specific physiological roles. Although functional interactions with the Na,K-ATPase have been clearly demonstrated, it is not known which domains and which residues interact with the alpha and/or beta subunits and affect the pump kinetics. The current study provides the first systematic analysis of structure-function relations of CHIF and gamma. It is demonstrated that the stability of detergent-solubilized complexes of CHIF and gamma with alpha and/or beta subunits is determined by the trans-membrane segments, especially three residues that may be involved in hydrophobic interactions. The transmembrane segments also determine the opposite effects of CHIF and gamma on the Na+ affinity of the pump, but the amino acids involved in this functional effect are different from those responsible for stable interactions with alpha.     

Concepts of the oligomeric structure and function of Na, K-ATPase based on the results of studying ligand binding and of determining the size of the molecular target

This review is devoted to the discussion of the sizes and molecular structure of the minimal functional unit of Na, K-ATPase. Special attention is paid to the data obtained by radiation inactivation method and studies on ligand binding. The model for the stepwise radiation inactivation of Na, K-ATPase is proposed. The conclusion is drawn that Na, K-ATPase has a dimeric structure, the interactions between its alpha-subunits stabilize the quaternary structure of the pump. Functionally, each alpha-subunit in a stabilized structure possesses a full hydrolytic activity.     

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.     

Dissociation of Na+,K+-ATPase inhibition from digitalis inotropy.

Recent findings from our laboratory as well as those of other laboratories do not support the postulation that the mechanism of the positive inotropic action of digitalis is due to inhibition of NA,K-ATPase. Using short-acting digitalis steroids and drug washout experiments, in isolated myocardial preparations, it has been demonstrated that Na,K-ATPase isolated from such preparations is still significantly inhibited, whereas the positive inotropic effect is no longer present. Also, based on kinetic measurements the two exponential rate constants observed for drug half-life, a rapid and slow phase, were found to be associated, respectively, with the very short inotropic half-life and the very long enzyme inhibition half-life. In addition, a dissociation of the transient inotropic effects of digitalis was observed from the long lasting cardiotoxic effects of digitalis during drug washout. Moreover, a temporal correlation was noted between the persistent inhibitory effects of digitalis on Na,K-ATPase and the persistent cardiotoxic effects of digitalis. Therefore, it is concluded that inhibition of Na,K-ATPase is not responsible for the positive inotropic action of digitalis, but may be the mechanism, at least in part, for certain cardiotoxic effects of digitalis.     

Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity. 2. Thermodynamics of kinetic cooperativity

The principles of structural kinetics allow one to define the thermodynamic conditions that are sufficient to generate a certain type of kinetic behavior. If subunits are loosely coupled, that is if no quaternary constraint exists between them, the kinetic behavior of the polymeric enzyme is qualitatively defined by the behavior of an ideal dimer. The nature and the extent of the kinetic cooperativity are defined by the energy of interaction, delta G rho, between two subunits. This energy of interaction is that of an ideal dimer relative to that of the A2 and B2 states. This thermodynamic formulation of a given type of cooperativity holds whatever the degree of polymerization of the enzyme. Under these conditions of loose coupling between subunits, positive kinetic cooperativity cannot be associated with any sigmoidicity of the rate curve. The range of energy coupling where positive kinetic cooperativity must, of necessity, be observed becomes more and more narrow as the number of subunit interactions is increased. This range, however, is independent of the number of subunits. The same situation is not observed for negative cooperativity which appears to be independent of both the number of subunits and the number of subunit interactions. If the subunits are tightly coupled, that is if quaternary constraints exist between them, three thermodynamic parameters, delta G' rho, delta G lambda, delta G mu, are required to define the nature of kinetic cooperativity. delta G' rho is the free energy of an ideal strained dimer relative to that of strained A2 and B2 states. delta G lambda and delta G mu represent the difference of strain energies between conformations A and B and B and B relative to that existing between conformations A and A. One may determine in the parametric space (delta G' rho, delta G lambda, delta G mu) the boundaries between the sufficient conditions that generate a certain type of cooperativity and the lack of these conditions. The kinetic parameters of the rate equation are not all independent. A number of constraint conditions exist between them which depend upon the subunit design of the polymeric enzyme. The existence of these constraint conditions may be diagnostic of a certain type of subunit interactions.     

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K- ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.     

Configuration of subunits within crystals of Na, K-ATPase maintained in the frozen-hydrated state

Two-dimensional crystalline sheets of Na, K-ATPase were studied in the vitrified, frozen-hydrated state by electron microscopy and image processing. The technique of correlation averaging was used to determine the projected structure. The projection map shows asymmetry between the pair of "alpha beta" protomers comprising a dimer of Na, K-ATPase molecules. The two protomers differ in overall density as well as in shape. One protomer has an oblong shape, whereas the other with higher density has a head and a hook region. Such an asymmetry has not been reported by other laboratories. This asymmetry may either be due to the coexistence of two different conformations of the enzyme in the dimeric form or due to the simultaneous existence of two molecular species of Na, K-ATPase.     

Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?

The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the alpha-subunit at the T(2) trypsin cleavage site were established. Using such conditions, the shark enzyme N-terminus was much more susceptible to proteolysis than the pig enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark enzyme, whereas it was significantly decreased for the pig kidney enzyme. The apparent ATP affinity was unchanged for shark but increased for pig enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E(1)-E(2) conformational equilibrium toward E(1). The phosphorylation and the main rate-limiting E(2) --> E(1) step are both accelerated after N-terminal truncation of the shark enzyme, but decreased significantly in the pig kidney enzyme. Some of the kinetic differences, like the acceleration of the phosphorylation reaction, following N-terminal truncation of the two preparations may be due to the fact that under the conditions used for N-terminal truncation, the C-terminal domain of the FXYD regulatory protein of the shark enzyme, PLMS or FXYD10, was also cleaved, whereas the gamma or FXYD2 of the pig enzyme was not. In the shark enzyme, N-terminal truncation of the alpha-subunit abolished association of exogenous PLMS with the alpha-subunit and the functional interactions were abrogated. Moreover, PKC phosphorylation of the preparation, which relieves PLMS inhibition of Na,K-ATPase activity, exposed the N-terminal trypsin cleavage site. It is suggested that PLMS interacts functionally with the N-terminus of the shark Na,K-ATPase to control the E(1)-E(2) conformational transition of the enzyme and that such interactions may be controlled by regulatory protein kinase phosphorylation of the N-terminus. Such interactions are likely in shark enzyme where PLMS has been demonstrated by cross-linking to associate with the Na,K-ATPase A-domain.     

Inhibition of Na,K-ATPase by a new ATP analog, adenosine-5-N'-(2,4-dinitro-5-fluorophenyl)phosphohydrazide.

A new ATP analog, adenosine-5-N'-(2,4-dinitro-5-fluorophenyl) phosphohydrazide (DNPH-AMP), has been synthesized, which is an irreversible inhibitor of Na,K-ATPase. Interaction of the analog with the enzyme in the presence of K+ is described by the scheme: [formula: see text] and corresponding kinetic constants k3 and Ki are found equal to 2.5 min-1 and 1.6 mM. In the presence of Na+ the time course of enzyme inactivation by DNPH-AMP is a biphasic curve in the semilogarithmic plot. The k3 and Ki values calculated for this case according to Fritzsch [Fritzsch (1985) J. Theor. Biol. 117, 397] are equal to 2.45 min-1 and 2.5 mM, respectively. ATP transforms the K(+)-type of Na,K-ATPase inactivation into the one that takes place in the presence of Na+.     

Mutational analysis of alpha-beta subunit interactions in the delivery of Na,K-ATPase heterodimers to the plasma membrane

The beta-subunit of the Na,K-ATPase is required to deliver functional alpha beta-heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the beta-subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the alpha-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of beta-subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by beta-subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated beta-subunits and some disulfide bridge substitution mutants assemble with the alpha-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.     

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.     

The use of biochemical solid-phase techniques in the study of alcohol dehydrogenase. 1. Some studies on subunit interactions in alcohol dehydrogenase with subunits covalently bound to agarose

The EE and SS isozymes of horse liver alcohol dehydrogenase have been immobilized separately to weakly CNBr-activated Sepharose 4B. The resulting immobilized dimeric preparations lost practically all of their activity after treatment with 6 M urea. However, enzyme activity was regenerated by allowing the urea-treated Sepharose-bound alcohol dehydrogenase to interact specifically with either soluble subunits of dissociated horse liver alcohol dehydrogenase or soluble dimeric enzyme. The regeneration of steroid activity in the immobilized preparations after treatment of the bound S subunits with soluble E subunits seems to show that true reassociation of the enzyme had taken place on the solid phase, since only isozymes with an S-polypeptide chain are active when using 5 beta-dihydrotestosterone as substrate. The results presented in this paper indicate that immobilized single subunits of horse liver alcohol dehydrogenase are inactive and that dimer formation is a prerequisite for the enzymic activity.     

Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits [Orhanovi? S., Pavela-Vrancic M. and Flogel-Mrsi? M. (1994) Acta. Pharm.44, 87-95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg2+ binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analyses. An activation study has indicated that Mg2+ enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn2+ or Mg2+ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release. APase displays higher activity in the presence of Mg2+, as binding of Mg2+ increases the rate of conformational change. A conformationally controlled and Mg2+-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of Pi into the environment.     

Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K(+) affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.     

Preparation of Na,K-ATPase specifically modified on the anti-fluorescein antibody-inaccessible site by fluorescein 5'-isothiocyanate

Specific labeling is required for energy transfer measurements and to avoid artifacts in the use of fluorophores as reporter groups. Therefore, a method for specific modification by one of the most popular reagents for P-type ATPases (fluorescein 5'-isothiocyanate) has been developed. Sulfhydryl reagents protected against modification of cysteine residues, and treatment with dithiothreitol eliminated a slow doubling of the fluorescence of conventionally modified Na,K-ATPase upon dilution that is attributed to disappearance of self-energy transfer. Removal of nonspecifically bound fluorescein was also confirmed by titration of the modified Na, K-ATPase with anti-fluorescein antibody and by time resolution of the fluorescence change when the modified enzyme was mixed with Na(+) in a stopped-flow instrument. The only fluorescence change when specifically modified Na,K-ATPase was mixed with Na(+) was the signal from fluorescein at the antibody-inaccessible, substrate-protectable site that reports the conformational change in unphosphorylated enzyme. The magnitude of the fluorescence change reporting the conformational change increased from between 8 and 12% to between 25 and 30% without affecting the kinetic constants estimated from titrations with Na(+) and K(+). The method should be generally applicable to the preparation of specifically labeled P-type pumps for use in kinetic and equilibrium titrations or energy transfer measurements.     

The molecular mechanism of the Na, K-ATPase system

Some new properties of Na,K-ATPase system have been revealed using the kinetic analysis of the complex enzymic systems. The fundamental mechanism of Na,K-ATPase functioning has been interpreted and the minimum model including all known working modes of the enzyme under different conditions has been built. The existence of new unknown modes and properties of Na,K-ATPase is predicted and confirmed by different authors.