©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Influence of Subunit Structure on the Interaction of Na/K-ATPase Complexes with Na
A CHIMERIC SUBUNIT REDUCES THE Na DEPENDENCE OF PHOSPHOENZYME FORMATION FROM ATP (*)

Kurt A. Eakle (1)(§), Rong-Ming Lyu (1), Robert A. Farley (1) (2)

From the (1) Department of Physiology and Biophysics and (2) Department of Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

High-affinity ouabain binding to Na/K-ATPase (sodium- and potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires phosphorylation of the subunit of the enzyme either by ATP or by inorganic phosphate. For the native enzyme (/1), the ATP-dependent reaction proceeds about 4-fold more slowly in the absence of Na than when saturating concentrations of Na are present. Hybrid pumps were formed from either the 1 or the 3 subunit isoforms of Na/K-ATPase and a chimeric subunit containing the transmembrane segment of the Na/K-ATPase 1 isoform and the external domain of the gastric H/K-ATPase subunit (/NH1 complexes). In the absence of Na, these complexes show a rate of ATP-dependent ouabain binding from 75-100% of the rate seen in the presence of Na depending on buffer conditions. Nonhydrolyzable nucleotides or treatment of ATP with apyrase abolishes ouabain binding, demonstrating that ouabain binding to /NH1 complexes requires phosphorylation of the protein. Buffer ions inhibit ouabain binding by /NH1 in the absence of Na rather than promote ouabain binding, indicating that they are not substituting for sodium ions in the phosphorylation reaction. The pH dependence of ATP-dependent ouabain binding in the presence or absence of Na is similar, suggesting that protons are probably not substituting for Na. Hybrid /NH1 pumps also show slightly higher apparent affinities (2-3-fold) for ATP, Na, and ouabain; however, these are not sufficient to account for the increase in ouabain binding in the absence of Na. In contrast to phosphoenzyme formation and ouabain binding by /NH1 complexes in the absence of Na, ATPase activity, measured as release of phosphate from ATP, requires Na. These data suggest that the transition from EP to EP during the catalytic cycle does not occur when the sodium binding sites are not occupied. Thus, the chimeric subunit reduces or eliminates the role of Na in phosphoenzyme formation from ATP, but Na binding or release by the enzyme is still required for ATP hydrolysis and release of phosphate.


INTRODUCTION

The Na/K-ATPase() is a membrane-embedded enzyme complex that utilizes energy derived from ATP hydrolysis to transport Na and K ions across cell membranes. The active enzyme complex consists of two dissimilar subunit proteins, an subunit of approximately 1000 amino acids, and a subunit of approximately 300 amino acids. The subunit of Na/K-ATPase is structurally and evolutionarily related to a large gene family of P-type ATPases which transport a number of different cations in organisms ranging from archaebacteria to mammals. Amino acids that participate in the binding of cardiac glycosides by Na/K-ATPase (1, 2, 3, 4, 5, 6) are located in the binding site for substrate ATP (7, 8) , or whose chemical modification inactivates cation occlusion (9, 10) , have been mapped to the subunit. The Na/K-ATPases and H/K-ATPases form a closely related subfamily of P-type ATPases that are unique in their requirement for a subunit. The role of the subunit in Na/K-ATPase or H/K-ATPase function is poorly understood. The formation of a complex between the and subunits is essential for enzyme activity (11, 12) , and there is evidence that complex formation occurs before and subunits are transported from the endoplasmic reticulum to the plasma membrane (13, 14) . Recently, new isoforms of the Na/K-ATPase subunit (2 or AMOG (15, 16) ) and the subunit for H/K-ATPase (HK (17, 18, 19) ) have been identified which show only 25-35% sequence identity with the original Na/K ATPase subunit isoform (1). Heterologous expression of these new isoforms in yeast and Xenopus oocytes has confirmed that they are also capable of assembling with Na/K-ATPase subunits into active enzyme complexes. Complexes with the HK isoform, however, show differences in complex stability (20) and require higher concentrations of K to stimulate ATP hydrolysis and ion transport (21, 22) . These results, together with results from chemical modification of the H/K ATPase (23) and Na/K-ATPase (24) , raise the possibility that the subunit participates in ion binding and active ion transport.

Because yeast do not have an endogenous Na/K-ATPase, expression of mammalian Na/K-ATPase in yeast provides the opportunity to test the functional properties of specific combinations of and isoforms of Na/K-ATPase. In an initial study, chimeric subunits were formed by combining the intracellular and transmembrane regions of the rat Na/K-ATPase 1 subunit and the extracellular region of the rat gastric H/K-ATPase subunit (NH1) or the intracellular and transmembrane regions of the rat gastric H/K-ATPase subunit and the extracellular region of the rat Na/K-ATPase 1 subunit (HN1) (20) . Co-expression of these chimeric subunits with either the sheep 1 or rat 3 subunits of Na/K-ATPase in yeast cells resulted in the formation of functional hybrid Na/K-ATPase complexes that bind ouabain with high affinity (K 5-15 nM). Ouabain binds with high affinity to a phosphoenzyme intermediate in the Na/K-ATPase reaction cycle formed from either ATP or inorganic phosphate (P) (25, 26) . High affinity ouabain binding, therefore, reflects the formation of active pump complexes. Ouabain binding to phosphoenzyme formed from Mg and ATP is stimulated by the presence of Na, whereas ouabain binding to phosphoenzyme formed from Mg and P is inhibited by either Na or K(27, 28, 29) . Mg- and P-dependent ouabain binding to complexes formed from and chimeric subunits was also inhibited by both K(20) and Na.() Structural features located in the external domain of the subunit appear to be responsible for differences in the apparent K affinity between Na/K-ATPase subunits and H/K-ATPase , whereas differences in the stability of /1 and /HK complexes appear to map to the cytoplasmic/transmembrane region of the subunit (20) .

In this study the role of Na in promoting phosphoenzyme formation and ouabain binding from ATP for various / combinations has been studied. Complexes formed with either 1 and 1 or the HK subunit show a similar dependence on Na for phosphorylation and ouabain binding. After a short (3 min) incubation, more ouabain is bound at low (1-2 mM) concentrations of Na by 3/HK complexes than by 3/1 complexes, suggesting that the rate of phosphoenzyme formation is faster in 3/HK complexes. In contrast, complexes of isoforms combined with the NH1 chimera were able to efficiently form phosphoenzyme from ATP and bind high levels of ouabain in the absence of Na. In this case, ouabain binding still required phosphoenzyme formation and was largely independent of buffer ions and pH, suggesting that other ions were not substituting for Na. However, although /NH1 complexes would bind ouabain in an ATP-dependent reaction in the absence of Na, overall ATP hydrolysis did not occur at measurable rates for /NH1 complexes in the absence of Na. Thus, although phosphoenzyme may form under these conditions, the enzyme still requires Na to complete the reaction cycle. These results show that the structure of the subunit can influence both the interaction of the enzyme with Na and the kinetics of Na/K-ATPase reactions.


EXPERIMENTAL PROCEDURES

Materials

The yeast strain 30-4 (MAT , trp1, ura3, Vn2, GAL) was obtained from R. Hitzeman (Genentech) and was used for all heterologous expression studies. [H]Ouabain (specific activity, 23-25 Ci/mmol) was purchased from DuPont NEN. cDNAs encoding the rat 3 and rat 1 subunits were obtained from Edward Benz (Yale), the sheep 1 cDNA was obtained from Jerry Lingrel (University of Cincinnati), and the rat HK cDNA was obtained from Robert Levenson (Yale). Clones of the sheep 1 cDNA in the vector YEp1PT (YEpNK), the rat 3 cDNA in the vector YEp1PT (YEpR3), the rat 1 cDNA in the vector pG1T (pG1T-R1), and the rat H/K-ATPase subunit in the vector pG1T (pG1T-HK) have been described previously (12, 21, 30) . Construction of chimeric subunits (NH1 and HN1) and cloning into the expression plasmid pG1T (yielding pG1T-NH1 and pG1T-HN1, respectively) have been described previously (20) .

Methods

Standard yeast media were used throughout this study (31) . The yeast strain 30-4 was transformed with different combinations of the subunit expression plasmids (YEpNK or YEpR3) and subunit expression plasmids (pG1T-R1, pG1T-HK, pG1T-NH1, or pG1T-HN1) using the LiAc procedure of Ito (32) . Following selection on minimal media for transformation with both plasmids, frozen glycerol stocks were made of transformants and were stored at -80 °C. Transformed colonies for the different + combinations were grown in YNB-galactose medium using a mixture of supplements (31) omitting tryptophan and uracil to maintain selection for the expression plasmids. A microsomal membrane fraction was isolated as described (21). Protein concentrations were assayed by the method of Lowry (33) . Mg- and P-dependent [H]Ouabain Binding-Assays for [H]ouabain binding were done in duplicate using (final concentrations) 20 nM [H]ouabain, 4 mM MgCl, 4 mM Tris PO, 50 mM Tris-HCl, pH 7.4, and between 0.25 and 1 mg of membrane protein/assay. Assays were rocked at 37 °C for 1 h, chilled on ice/HO for 15 min, and pelleted in a microcentrifuge (Eppendorf) for 15 min at 4 °C. The tubes were rinsed briefly with 0.5 ml of ice-cold buffer, and pellets were suspended with 1% SDS prior to scintillation counting. Nonspecific binding was determined in duplicate by the addition of 1 mM non-radioactive ouabain and was subtracted from assay values. A mock-assay was performed without [H]ouabain, and the pellet was solubilized in 1% SDS and was assayed for protein recovery. Membranes from untransformed yeast or yeast transformed with vectors alone showed [H]ouabain binding equal to the nonspecific binding measured with the addition of 1 mM non-radioactive ouabain, typically 20 fmol of [H]ouabain/mg of microsomal protein.

ATP-dependent Ouabain Binding

Microsomal membranes from yeast expressing the desired / combinations were washed free of Na by diluting the membranes about 25-fold in either sodium-free buffer (25 mM Imidazole, 1 mM EDTA (free acid), pH 7.4) or distilled water, and membranes were pelleted for 60 min 4 °C at 50,000 rpm in a Beckman Ti-70 rotor. The membrane pellet was suspended in sodium-free buffer or distilled water and was pelleted again before being suspended in a small volume of buffer or distilled water. Microsomal membranes and reaction tubes for ATP-dependent ouabain binding were prewarmed for 10 min at 37 °C, and the reaction was initiated by the addition of membranes. Reactions were incubated for 3 min at 37 °C and were stopped by transfer to ice/HO. Bound [H]ouabain was separated from free [H]ouabain by centrifugation for 15 min, 4 °C in a microcentrifuge (Eppendorf), and aspiration of the supernatant. The reaction tubes were rinsed with 0.5 ml of ice-cold distilled water, and pellets were suspended in 200 µl of 1% SDS prior to scintillation counting. Final concentrations in the total reaction volume were 20 nM [H]ouabain, 5 mM MgCl, and appropriate concentrations of buffer (typically 35 mM imidazole, pH 7.4), ATP (Tris salt, typically 100 µM) (Sigma), and NaCl as indicated. Mg- and P-dependent ouabain binding was performed on washed membranes at the same time as ATP-dependent ouabain binding in order to estimate the total number of ouabain binding sites available and the protein recovery during separation of bound and free [H]ouabain. Protein recovery was typically around 70%. Assays were done in duplicate and nonspecific binding, determined by the addition of 1 mM unlabeled ouabain, was subtracted from assay values. Stock solutions of 10 or greater concentrations of all reagents and washed membranes were assayed by flame photometry for Na. In all cases, the reagents failed to register a measurable amount of Na, although the sensitivity of the instrument was 1 mM Na minimum.

For experiments with AMP-PNP, the tetra-lithium salt of AMP-PNP (Sigma) was used. Preliminary experiments suggested that the solution was contaminated with 0.5% ATP, so 1 ml of a 0.5 mM AMP-PNP solution was treated with 50 units of apyrase (Sigma) for 30 min at 30 °C before use. A similar solution of ATP was also treated with apyrase as a control for complete conversion of ATP to AMP.

Measurement of ATPase Activity by PRelease

Yeast microsomal membranes were extracted with SDS (34) as described previously (20) . Membranes were washed free of Na as described above and were assayed for ATPase activity by phosphate release (7, 8) . Sodium concentrations were as indicated in the figures, and potassium was 10 mM in all assays. Each reaction contained 80 µg of yeast membrane protein and was incubated at 37 °C for 30 min.

Data Analysis

For titrations with ATP, the measured levels of [H]ouabain bound were plotted against the increasing concentrations of ATP added. Data were fit by a first order reaction model [Bound = Bound [ATP]/(K + [ATP]) + C] using the nonlinear curve fitting function (Levenberg-Marquardt algorithm) of Slidewrite Plus for Windows (Advanced Graphics Software, Inc., Carlsbad, CA). The constant term was added to the equation to account for the small amount of ATP-independent binding that was observed in the reaction in the absence of ATP. As shown in Fig. 2and 3, this binding could largely be attributed to Mg+P-dependent binding. For the Na dependence of ATP hydrolysis, measured levels of P released (expressed as percent of maximal activity) were plotted against increasing concentrations of Na. Data were fit by the Hill equation for multisite reaction mechanisms [% activity = 100 [Na] /{[Na]+ (K)}], using the method described above to give estimates of the K for Na and the Hill coefficient n.


Figure 2: ATP-dependent [H]ouabain binding by 1/1 and 1/NH1. Microsomal membranes from yeast expressing the indicated / subunits were washed free of Na and assayed for ATP-dependent [H]ouabain binding in 35 mM imidazole/HCl, pH 7.4, 5 mM MgCl with or without 100 µM Tris-ATP and with or without 10 mM NaCl as indicated in the figure. Nonspecific binding in the presence of 1 mM ouabain was subtracted from all values. Bars indicate the average of duplicate points and the variation between duplicates, including variation in the nonspecific binding. The percent specific binding for each condition, normalized to 50 µM ATP, 10 mM Na as 100%, is indicated under each bar.




RESULTS

ATP-dependent Binding of [H]Ouabain-During the transport of Na and K, Na/K-ATPase undergoes a reaction sequence that includes Na binding at the intracellular face of the enzyme, ATP hydrolysis, Na transport across the plasma membrane, K binding at the extracellular face of the enzyme, and its transport to the cytoplasm of the cell (Fig. 1A) (35, 36) . Ion translocation is thought to occur during transitions between two major conformations of the enzyme (E1 and E2). Additional conformational intermediates have been inferred from the sensitivity of the phosphoenzyme to ADP and K(26, 37) . The cardiac glycoside ouabain binds with high affinity to Na/K-ATPase when it is phosphorylated and is in the E*P conformation (26) . Little or no ouabain binding is detectable in the absence of phosphorylation indicating that the affinity of nonphosphorylated conformations of the pump for ouabain is much lower than the E*P conformation. Under normal physiological conditions, sodium binding to the enzyme promotes the efficient formation of phosphoenzyme from ATP (``front door'' phosphorylation), and subsequent ouabain binding. Phosphoenzyme may also be formed by incubating the enzyme with Mg and P (``back door'' phosphorylation; Fig. 1B). In this case, the addition of Na to the reaction will drive the enzyme toward the E1 conformation and will inhibit ouabain binding. Mg is a required co-factor in both the front door and back door phosphorylation reactions. Since the hydrolysis of ATP leads to release of P, phosphoenzyme formation by either reaction is possible under front door conditions.


Figure 1: Schematic representation of the reaction cycle of Na/K-ATPase and the interaction with ouabain. (A) reaction scheme for ATP-dependent Na and K transport by Na/K-ATPase. Adapted from Ref. 39, with modifications based on Ref. 26. B, reaction scheme for Mg + P-dependent ouabain binding by Na/K-ATPase.



Fig. 2 shows ouabain binding to either 1/1 (A) or 1/NH1 (B) under front door phosphorylation conditions. A membrane fraction from yeast cells expressing the indicated subunits was washed free of Na by a 25-fold dilution in Na-free buffer and sedimentation in an ultracentrifuge twice before being resuspended in Na-free buffer. The sodium concentration in all reagents was below the detection limit of flame photometry. Membrane samples were incubated in the presence of 20 nM [H]ouabain, 5 mM Mg, and in the absence or presence of 100 µM Tris-ATP and/or 10 mM Na as indicated. The reaction was allowed to proceed for 3 min in order to ensure that ATP was not depleted and that a significant level of P, which would lead to back door ouabain binding, was not released. After the 3-min incubation, membranes were collected by centrifugation.

As shown in A, incubation of 1/1 with ATP in the presence of Na resulted in the highest amount of ouabain bound in 3 min. The amount of ouabain bound in 3 min under these conditions was 25% of the total ouabain binding capacity of these membranes as determined in an equilibrium binding assay (). In the absence of ATP and Na, ouabain binding to 1/1 was only 5.3% of the amount bound in the presence of ATP and Na (1.3% of total binding), indicating that ouabain binding in this assay required the presence of ATP. The addition of Na in the absence of ATP reduced ouabain binding almost to zero. This indicates that, in the absence of ATP, ouabain was binding was to phosphoenzyme formed from P, since Na will compete in the back door reaction for phosphoenzyme formation and ouabain binding. Measurements of P in yeast membranes incubated similarly without ATP showed that 2 µM P is present in the samples, consistent with this suggestion.() This phosphate is likely to be derived from the breakdown of membrane phospholipids. Incubation of 1/1 complexes for 3 min with ATP in the absence of Na resulted in 36% of the amount of ouabain bound compared with ATP + Na conditions. While some of this can be accounted for by a small amount of back door phosphorylation and ouabain binding (5% of ATP + Na), there is clearly some phosphorylation and ouabain binding that is ATP dependent even in the absence of Na. Since formation of the phosphoenzyme is the rate-limiting step in ouabain binding (38, 39) , this indicates that Na is not required for ATP-dependent phosphorylation. Instead, for normal Na/K ATPase complexes of 1/1, the formation of phosphoenzyme from ATP in the absence of Na proceeds more slowly than in the presence of Na. Control experiments show that when dog kidney microsomes containing 1/1 are used, maximal binding is observed with ATP + Na, whereas in the absence of Na about 35% of the ouabain binding seen with ATP + Na is observed (data not shown). This indicates that changes caused by expression of 1/1 in yeast such as differences in glycosylation or an altered phospholipid content of the membranes are not responsible for these observations.

When the same measurements were made using 1/NH1 (Fig. 2B), maximum ouabain binding after 3 min was also observed in the presence of ATP and Na. In this instance, 33% of the total ouabain binding capacity was reached after 3 min (). Like with 1/1, a small amount of ouabain was bound to 1/NH1 in the absence of ATP (19.5% of the amount bound in the presence of Na and ATP; 6.5% of total sites), and this was reduced in the presence of Na (4.2% of ATP + Na; 1.4% of total sites), suggesting some back door phosphorylation of the pump. In contrast to 1/1, however, incubation of 1/NH1 with ATP in the absence of Na resulted in a relatively high level of ouabain binding after 3 min (74% of ATP + Na). If ouabain binding by 1/NH1 complexes is limited by the formation of phosphoenzyme as it is for 1/1 complexes, then this result suggests that phosphorylation of 1/NH1 from ATP in the absence of Na proceeds more rapidly than phosphorylation of 1/1 under the same conditions. As shown below, similar results were also obtained for the 3 pump complexes, indicating that the NH1 subunit has a general effect of increasing the rate of phosphoenzyme formation from ATP in the absence of Na.

Phosphorylation Is Required for ATP-dependent Ouabain Binding to /NH1 Complexes

The high level of ATP-dependent ouabain binding to /NH1 complexes in the absence of Na might be explained if ATP hydrolysis and phosphoenzyme formation were no longer required for high affinity ouabain binding. This could occur, for example, if nucleotide binding were sufficient for the hybrid pump to fold into a conformation that resembles the phosphoenzyme. In the experiment shown in Fig. 3, ouabain binding to 3/1 or 3/NH1 complexes was measured after a 3-min incubation with either 0 or 10 mM added Na in the presence of either 50 µM ATP, no ATP or 50 µM AMP-PNP. As shown in Fig. 3A, ouabain binding to 3/1 complexes was highest in the presence of ATP and 10 mM Na, as previously observed for 1 complexes. Similar to the results with 1/1, 3/1 complexes gave only a low level of ouabain binding in the presence of ATP and the absence of Na (13% of ATP + Na). Deleting ATP from the reaction resulted in only low levels of ouabain binding in either the absence of Na (9% of ATP + Na) or in the presence of 10 mM Na (4% of ATP + Na). Incubation of 3/1 in the presence of the nonhydrolyzable ATP analog AMP-PNP gave only low levels of ouabain binding similar to those seen in the absence of ATP. If ATP was hydrolyzed to AMP and P by incubation with apyrase before the reaction, a low level of ouabain binding was observed that was identical to samples incubated with the same concentration of P for 3 min (data not shown). This confirms that ouabain binding from ATP requires a phosphate that can be transferred from the nucleotide to the enzyme.


Figure 3: Phosphoenzyme formation is required for front door ouabain binding. Front door ouabain binding to 3/1 (A) or 3/NH1 (B) was done for 3 min in the presence of 50 µM Tris ATP (+ATP), without ATP (-ATP), or 50 µM AMP-PNP (AMP-PNP). Assays were done in duplicate either in the absence of Na or the presence of 10 mM NaCl as indicated in the figure. Bars represent the mean of duplicate values with error bars indicating the variation. The percent specific binding for each condition, normalized to 50 µM ATP, 10 mM Na as 100%, is indicated under each bar.



A similar dependence of ouabain binding on ATP was observed for 3/NH1 complexes (Fig. 3B). The maximum amount of ouabain was bound after 3 min in the presence of ATP and Na. However, a high level of ouabain binding by 3/NH1 (68% of ATP + Na) was also observed in the presence of ATP and the absence of Na, similar to the results in Fig. 2 for 1/NH1. In the absence of ATP, only 7-14% of the amount of ouabain bound in the presence of ATP and Na was observed. As with 3/1, ouabain binding in the absence of ATP was inhibited by the addition of Na, suggesting that it is due to back door phosphorylation of the pump. Substitution of AMP-PNP for ATP resulted in only low levels of ouabain bound by 3/NH1, similar to samples with no ATP added. Likewise, if ATP was hydrolyzed to AMP and P by incubation with apyrase before the assay, ouabain binding was inhibited by the addition of 10 mM Na in an identical fashion to samples incubated with the same concentration of P for 3 min (data not shown). This indicates that nucleotide binding to /NH1 complexes alone is not sufficient to support ouabain binding in this assay. These results indicate that high-affinity ouabain binding by complexes formed between Na/K-ATPase subunits and either the native 1 subunit or the chimeric NH1 subunit requires the hydrolysis of ATP and the formation of a phosphoenzyme intermediate.

It is interesting to note that /NH1 complexes show more ouabain binding over a short time course than /1 complexes. This is seen with both 1 and 3 isoforms, and with both front door and back door phosphorylation conditions. In , the amounts of ouabain bound from either (ATP + Na) or P in 3 min are compared with the total amount of ouabain binding sites present in the same membranes. For 1/1, front door phosphorylation (ATP + Na) results in 25% of the total ouabain binding sites occupied in a 3-min assay, whereas in the same conditions 33% of 1/NH1 sites will bind ouabain. Likewise, ouabain will bind to 40% of 3/1 sites in 3 min from ATP, whereas 45% of 3/NH1 sites will be occupied in the same time. When 100 µM P is used to form phosphoenzyme, a smaller fraction of sites are occupied in 3 min, indicating that phosphorylation from P is kinetically slower than phosphorylation from ATP. However, complexes of /NH1 still show more ouabain binding from P under these conditions than complexes of /1. In the case of 1, 1/1 complexes show 9% of total sites occupied in 3 min, whereas 1/NH1 complexes show 15%. Complexes of 3 show a similar increase from 9 (3/1) to 22% (3/NH1) of total sites occupied. Although the effect is small in magnitude, our experiments consistently show that the presence of the NH1 subunit increases the amount of ouabain binding seen in a short time course. Since the formation of the phosphoenzyme is the rate-limiting step in ouabain binding, this suggests that the NH1 subunit slightly increases the rate of phosphoenzyme formation from either ATP or P.

The Role of Buffer Ions in Na-independent Ouabain Binding by /NH1

Certain buffer ions have been reported to have Na-like effects on the conformation of Na/K-ATPase (40, 41) . Schuurmans Stekhoven et al.(40, 41) have reported that at low concentrations of Mg (0.1 mM), imidazolium ion will substitute for Na in promoting the formation of phosphoenzyme from ATP by Na/K-ATPase. This effect is specific to low concentrations of Mg and is nearly eliminated at 2.5 mM Mg. Substitution of Tris for imidazolium ion does not support ATP-dependent phosphorylation of the pump. Because the effect of imidazole is increased at higher concentrations and is specific for the imidazolium ion, it was suggested that imidazolium ion may substitute for Na in the phosphorylation reaction. Fig. 4 shows the results of an experiment to test whether the substitution of buffer ions for Na ions could explain the increased binding of ouabain to /NH1 complexes in the absence of sodium. In this experiment, membranes were washed by centrifugation in distilled water to remove Na and buffer ions. For 3/1 (A), less than 30% of maximal ouabain binding was observed after 3 min in the absence of Na and in the presence of either 5, 20, or 50 mM Tris or imidazole. A similar result was observed (40% maximal binding) if buffer ions were excluded from the reaction, except for a small amount of Tris from the 100 µM Tris-ATP solution used. For 3/NH1 (B) the same amount of ATP-dependent ouabain binding was observed in unbuffered water in the presence or absence of 10 mM Na. Buffer ions do have some effects on the reaction, with higher amounts of ouabain bound in the presence of Na at low buffer concentrations compared with water and less ouabain bound in the absence of Na. Compared with 3/1, however, the 3/NH1 complex still binds 2-3-fold more ouabain in the absence of Na under all buffer conditions tested. Although a small amount of Tris could not be eliminated from the reaction, increasing the concentration of Tris inhibits ouabain binding in the absence or presence of Na. Thus, it does not appear that the increased level of ATP-dependent ouabain binding by /NH1 complexes in the absence of Na is due to substitution of buffer ions for Na.


Figure 4: The effects of buffer ions on ATP-dependent [H]ouabain binding. Microsomal membranes from yeast expressing the indicated / subunits were washed free of Na using distilled water and were assayed for front door [H]ouabain binding in either the absence of buffer (HO) or in the buffer indicated (pH 7.4) at concentrations of either 5, 20, or 50 mM, with 100 µM Tris-ATP and with 10 mM NaCl (solid bars) or without Na (open bars) as indicated in the figure. A shows binding to 3/1 complexes, and B shows binding to 3/NH1 complexes. Variation between duplicates was <10 fmol of ouabain bound in all cases (usually <2 fmol). The percent numbers on the open bars indicate the fraction of binding in the absence of Na relative to the binding observed for the same buffer conditions in the presence of 10 mM Na.



NaTitration of ATP-dependent Ouabain Binding

In the experiment shown in Fig. 5, the Na requirement for ATP-dependent ouabain binding by Na/K-ATPase complexes of 1, 3, and the 1, HK, NH1, and HN1 subunits was determined. The combination 3/HN1 appears to either assemble poorly or form an unstable complex when expressed in yeast and gives levels of ouabain binding which are too low to test reliably (20) . As shown in Fig. 5, the amount of ouabain bound by the combinations 1/1, 1/HK, and 3/1 increases as [Na] is increased, with half-maximum ouabain bound at 1-2 mM Na for 1/1 or 1/HK or at 3-4 mM for 3/1. For 3/HK, a 2-fold increase in the amount of ouabain binding is observed in the range of 1-2 mM Na, compared with 3/1. In all four cases, approximately 20-30% of maximal ouabain binding is observed in the absence of Na, again suggesting that the ATP-dependent phosphorylation and ouabain binding can proceed to a limited extent in the absence of Na. For 1/NH1 and 3/NH1, 65-75% of maximum ouabain binding is observed in the absence of Na. Addition of Na increases the amount of ouabain bound, with a K of approximately 0.6-0.8 mM. These results indicate that the /NH1 complexes have only a slightly higher apparent affinity for Na than /1 complexes. One possible explanation for high levels of ATP-dependent ouabain binding by /NH1 in the absence of Na would be if the /NH1 complexes have a high affinity for Na such that trace levels of contaminating Na in the reaction would be sufficient to promote phosphorylation and ouabain binding. However, addition of 50-200 µM Na to the reaction results in little or no increase in ouabain binding consistent with the absence of a very high affinity Na site on the /NH1 complexes.


Figure 5: [Na] dependence of ATP-dependent ouabain binding. Microsomal membranes were washed with Na-free imidazole buffer and assayed in the front door ouabain binding assay with increasing concentrations of NaCl. A shows the 1 subunit isoform combined with different subunits. B shows the 3 subunit isoform combined with different subunits. Duplicate points for each concentration of Na are shown and lines connect the mean values at each concentration. The leftmost point in each assay shows binding in the absence of added Na. Nonspecific binding was subtracted from each data set, and data are expressed as the percent maximum for each / combination.



Na titration of ATP-dependent ouabain binding by 1/HN1 complexes shows a behavior complementary to that of the 1/NH1 complexes. Here, ouabain binding in the absence of Na is less than the 1/1 complexes, and the K for Na has increased to approximately 10 mM. Thus, although the NH1 chimera seems to decrease the Na requirement for phosphoenzyme formation from ATP and increase the apparent Na affinity, the complementary subunit chimera HN1 seems to increase the Na requirement for phosphoenzyme formation from ATP and decrease the apparent Na affinity.

pH Dependence of Front Door Ouabain Binding

Protons have been reported to serve as substitutes for Na ions in Na/K-ATPase reactions (42, 43) . In order to examine whether protons are substituting for sodium ions in the ATP-dependent binding of ouabain to /NH1 complexes, the pH dependence of this reaction was examined (Fig. 6). The amount of ouabain bound by both 3/NH1 complexes and 3/1 complexes decreased as the pH was increased from 6.8 to 8.0 in the presence of 10 mM Na. In the absence of Na, ouabain binding by 3/NH1 complexes decreased from 71% of control in the presence of 10 mM Na at pH 6.8 to 55% of control at pH 8.0. The 3/1 complexes also show a small decrease in fractional ouabain binding from 32 to 26% as pH was increased from pH 6.8 to 8.0. The reduction in ATP-dependent ouabain binding as pH was increased is consistent with the substitution of protons for Na in the phosphorylation reaction, however, the magnitude of this reduction binding is small compared with the change in pH which represents more than an order of magnitude change in [H] from pH 6.8 to pH 8.0. Moreover, the pH-dependent change in ouabain binding in the presence of 10 mM Na is at least as great as the the changes observed in the absence of Na. The pH dependence of ouabain binding is similar to the pH dependence of phosphoenzyme formation by Na/K-ATPase reported by Forbush and Klodos (44) . This suggests that protons are playing a similar role for both the normal /1 complexes and /NH1 complexes. Nevertheless, it is hard to rule out a role for H in this reaction entirely since a change in pH can also affect the ionization state of charged amino acid side chains that may participate in cation binding and/or transport.


Figure 6: Effects of pH on ATP-dependent ouabain binding. Microsomal membranes with 3/1 (left panel) or 3/NH1 complexes (right panel) were washed with distilled water and were assayed in the front door ouabain binding assay using 25 mM imidazole buffer at the pH values indicated in the figure. Solid bars indicate binding done in the presence of 10 mM NaCl, and open bars indicate binding in the absence of added Na. Variation between duplicates was <20 fmol [H]ouabain bound in all cases (usually <5 fmol). The percentage on the open bars indicates the fraction of binding in the absence of Na relative to the binding observed for the same buffer conditions in the presence of 10 mM Na.



The Effect of the NH1 Subunit on Ouabain Affinity

Scatchard analysis of equilibrium [H]ouabain binding to both 3/1 and 3/NH1 complexes indicates that the pumps assembled with the chimeric subunit had about a 2-fold higher affinity for ouabain than the native pumps (data not shown). The K for ouabain binding to 3/1 complexes was 15.2 ± 4.1 nM (n = 4; ± S.D.), whereas the K for ouabain binding to 3/NH1 complexes was 6.7 ± 1.4 nM (n = 3; ± S.D.). A similar increase in affinity for ouabain has previously been observed when Na/K-ATPase subunits are assembled with the gastric H/K-ATPase subunit (21) .

Affinity of Pump Complexes for ATP

In Fig. 7 , the apparent affinity of both 1/NH1 complexes and 1/1 complexes for ATP was estimated by measuring the ATP concentration dependence of ouabain binding in the absence and presence of Na. For 1/1 complexes, the total amount of ouabain binding increases about 4-fold in the presence of added sodium, whereas for 1/NH1 complexes there is only a slight increase in total binding, similar to previous results. The apparent affinity (K) of 1/1 complexes for ATP in the absence of Na was 1.8 µM, and this increased to 0.8 µM with the addition of 10 mM Na. The apparent affinity of 1/NH1 complexes for ATP in the absence of Na was 1.3 µM, and this increased to 0.3 µM with the addition of 10 mM Na. Thus, under equivalent conditions, 1/NH1 complexes have a slightly higher apparent affinity for ATP than 1/1 complexes. The concentrations of ATP used in the experiments described in this report (50-100 µM ATP) are sufficient to saturate ATP binding both in the presence and absence of Na for both /1 and /NH1 complexes. Thus, although /NH1 complexes have a higher affinity for ATP, this increase in affinity is not sufficient to account for the increased level of ouabain binding observed in the absence of Na. The differences in affinity for ATP between /1 and /NH1 complexes are another example of the influence of subunit structure on enzyme properties.


Figure 7: Titration of ATP requirement for front door ouabain binding. Microsomal membranes with 1/1 (A) or 1/NH1 complexes (B) were washed with Na-free buffer and assayed in the front door ouabain binding assay with increasing concentrations of Tris-ATP. The + symbols indicate titrations done in the absence of added Na and the symbols indicate titrations done in the presence of 10 mM NaCl. Curves drawn show the fit to the data of a single site reaction model with a constant term added to account for ATP-independent ouabain binding in the reaction. The calculated constant values subtracted were 10.4 fmol (1/1, 0 Na), 13.2 fmol (1/1, 10 Na), 22.8 fmol (1/NH1, 0 Na), and 4.4 fmol (1/NH1, 10 Na). Correlation coefficients (r) for the data fits were 0.99 for 1/1, at 0 and 10 Na, and for 1/NH1 at 10 Na, and 0.96 for 1/NH1 at 0 Na. The apparent affinities for ATP in the reactions are indicated in the figure.



ATP Hydrolysis by /NH1 Complexes Requires Sodium

The high level of phosphoenzyme formation and ouabain binding by /NH1 complexes in the absence of Na suggests that hydrolysis of ATP by these complexes may no longer be Na-dependent and raises the possibility that ATP hydrolysis is not coupled to Na transport in these complexes. To examine this, the Na concentration dependence of ATP hydrolysis by different pump complexes was determined in an assay that measures release of P. In Fig. 8, the ATPase activities of 1/1 and 1/NH1 complexes (A) or 3/1 and 3/NH1 complexes (B) are plotted as a function of Na concentration. For all of the / combinations, there was no measurable ATPase activity in the absence of Na. Thus, although /NH1 complexes are able to form phosphoenzyme and bind ouabain in the absence of Na, they are unable to proceed through the reaction cycle and release phosphate at measurable rates in the absence of Na. The data for each / combination were normalized to the total number of Na/K-ATPase complexes determined by equilibrium ouabain binding in order to calculate the turnover number for each complex, and the Na concentration dependence was fit by the Hill equation in order to obtain the apparent affinities of each complex for Na and the Hill coefficients for the reaction (). For 1/1 and 3/1 complexes, the maximum turnover rate was 8000 ATP/min. Complexes of 1/NH1 and 3/NH1 were also able to hydrolyze ATP at maximum rates between about 6000 and 14,000 ATP/min. This indicates that under optimal conditions, /NH1 complexes are efficient ATPases and that the overall ability of the enzyme to proceed through the reaction cycle has not been severely compromised by the structural change in the subunit. The apparent affinity (K) of 1/1 and 3/1 complexes for Na in this reaction is 9.3 and 14.8 mM, respectively (). These values are higher than K values reported by Jewell and Lingrel (45) for modified rat 1 (1.2 mM) and 3 (3.1 mM) isoforms expressed in HeLa cells, although the higher apparent affinity of 1 than 3 for Na that was reported there is also seen here. The reasons for the quantitative differences in K are not known. Both 1/NH1 and 3/NH1 show an approximately 3-fold increase in apparent affinity for Na, when compared with 1/1 and 3/1 complexes, with K values of 3.6 and 4.4 mM, respectively. The apparent affinity for Na measured in this reaction reflects the interaction of Na with the enzyme at multiple reaction steps, and it is not possible using these data to determine which Na-dependent step(s) in the reaction cycle have been affected.


Figure 8: Na dependence of ATP hydrolysis. Yeast microsomal membranes containing either 1/1, 1/NH1, 3/1, or 3/NH1 pump complexes were washed by centrifugation to remove Na. Activity measurements were carried out at 37 °C for 30 min in the presence of 10 mM KCl, 5 mM MgCl, 3 mM Tris-ATP, 15 mM Tris-azide, 1 mM NaEDTA, and 50 mM Tris/HCl, pH 7.4. The concentration of NaCl was varied between 0 and 100 mM as indicated. Data for 50 mM NaCl are not shown. Each data point represents at least three experimental values obtained in duplicate, and the average values (±S.E.) are presented as the percentage of maximum Na/K-ATPase activity calculated after subtraction of ATPase activity measured in the presence of 1 mM ouabain. Curves are drawn to show the fit to the data by a highly cooperative Hill equation: [% maximum activity = 100 [Na]/{[Na] + (K)}]. A, 1/1 () and 1/NH1 (▾); B, 3/1 () and 3/NH1 (). Kinetic constants for each complex are shown in Table II.



Complexes of either 1/1 or 3/1 have Hill coefficients for the sodium dependence of ATP hydrolysis of 2.0 and 2.3, respectively (). When the NH1 subunit is substituted for 1, the 1/NH1 and 3/NH1 complexes show a decrease in the Hill coefficients to 1.0 and 1.3, respectively. This decrease suggests either that the stoichiometry of Na binding and transport has been altered by the NH1 subunit or that the extent of cooperativity of Na binding has been altered in these complexes.


DISCUSSION

The subunit of Na/K-ATPase shows extensive homology to the larger family of P-type cation transport ATPases, and numerous studies have identified sites within the subunit that are involved in ATP binding and hydrolysis. The role of the subunit as the catalytic subunit of the enzyme is clear. The role of the subunit, which is unique to the subfamily of Na/K-ATPase isoforms and the H/K-ATPase is poorly understood. Formation of a complex between the and subunits is required for Na/K-ATPase to exhibit Na- and K-stimulated ATP hydrolysis, high affinity ouabain binding, and cation transport activities (11, 12) . Assembly of complexes between Na/K-ATPase subunit isoforms and different isoforms of the Na/K-ATPase subunit or a subunit from the gastric H/K-ATPase in heterologous expression systems has made it possible to examine the effect of different subunit structures on Na/K-ATPase activity and to infer whether the subunit may contribute to enzymatic activity. When the H/K-ATPase subunit is substituted for the Na/K-ATPase 1 isoform, hybrid pumps are formed which show high affinity ouabain binding and Na and K transport (21, 22, 46) . Hybrid pumps of Na/K-ATPase 1 or 3 subunits and the H/K-ATPase subunit are more sensitive to extraction by SDS than 1/1 or 3/1 complexes (20) , suggesting that they form less stable complexes. In addition, 1/HK and 3/HK complexes require higher concentrations of K to inhibit ouabain binding or to stimulate ATPase activity, suggesting that the HK subunit lowers the affinity of the pump complexes for K(20, 21, 22) . A similar effect on K affinity has been reported for the 3 isoform from Bufo marinus(47) . Chimeric polypeptides between the 1 and HK isoforms indicate that the stability of / complexes depends at least in part on the transmembrane region of the subunit, since subunits containing the transmembrane region of the HK subunit either form less stable complexes or do not assemble with subunits (20) . For K affinity, the results are less clear. In one case, a chimera with the external domain of the HK subunit expressed in yeast seems to show the same effect on K affinity as seen with the whole HK subunit (20) , whereas a similar chimera expressed in Xenopus oocytes shows behavior intermediate between the 1 and HK controls (22) . These differences may be due to the fact that and isoforms from different species were used or to difficulties in distinguishing between endogenous Na/K-ATPase activity and the activity of heterologously expressed subunits in Xenopus oocytes.

In the results reported here, effects of differences in subunit structure on the interaction of Na/K-ATPase complexes with Na have been examined. For complexes of normal sodium pump isoforms (1/1 and 3/1), Na promotes the formation of a phosphoenzyme from ATP leading to high affinity ouabain binding. Low levels of ATP-dependent phosphorylation and ouabain binding occur in these complexes in the absence of Na ( Fig. 2and Fig. 3). This indicates that Na, while not absolutely required for ATP hydrolysis and phosphorylation, accelerates these reactions. An increase in the rate of ATP-dependent phosphoenzyme formation by Na has been also been reported for Na/K-ATPase in Torpedo electric organ (29) . It is possible that differences in the makeup of membrane lipids in yeast membranes contribute to this effect; however, control experiments with dog kidney microsomes show a slow rate of ATP dependent ouabain binding in the absence of Na which is accelerated when 10 mM Na is added (data not shown). These studies indicate that this observation is not unique to Na/K-ATPase expressed in yeast cells. The substitution of the HK subunit for 1 has no effect on the Na dependence of ouabain binding when combined with the 1 subunit; however, when HK is combined with the 3 subunit, this complex shows an increased level of ouabain binding in the presence of 1-2 mM Na (Fig. 5). Since phosphoenzyme formation is rate-limiting for ouabain binding (38, 39) , it is likely that the increased level of ouabain binding by 3/HK at low [Na] is due to an increase in the rate of phosphoenzyme formation.

Complexes of both 1 or 3 and the chimeric subunit NH1 show a dramatic increase in the level of ATP-dependent ouabain binding after 3 min in the absence of Na, exhibiting from 70-100% of maximum binding depending on buffer conditions. (Figs. 2-5). Phosphorylation from ATP is still required by /NH1 complexes for high-affinity ouabain binding, since AMP-PNP will not support front door ouabain binding (Fig. 3). Buffer ions such as Tris or imidazolium inhibit the reaction in the absence of Na, making it unlikely that they are substituting for Na. Changing the pH of the reaction from 6.8 to 8.0 results in a small decrease in the relative amount of binding in the absence of Na; however, this decrease is similar to the pH-dependent decrease in the presence of 10 mM Na. These results suggest that the differences observed are due to more general effects of pH on the reaction rather than the substitution of H for Na. The most likely explanation for the high levels of ouabain binding by /NH1 complexes in the absence of Na is the rapid formation of a phosphoenzyme under these conditions. In the absence of Na, /1 complexes are also able to form a phosphoenzyme from ATP and bind ouabain, and Na accelerates this reaction. It is possible that the /NH1 complexes adopt a conformation in the absence of Na similar to the conformation of /1 complexes when Na is bound and that the phosphorylation reaction proceeds by a similar mechanism in both cases. It also appears that phosphoenzyme formation is faster for /NH1 complexes not only from ATP, but also from P (). The increased rate of phosphorylation combined with the reduced role of Na in phosphoenzyme formation by /NH1 complexes raises the possibility that there may be subtle changes in the mechanism of ATP hydrolysis and phosphoenzyme formation.

Measurement of ATPase activity by ouabain-sensitive phosphate release from ATP shows that the maximum turnover of ATP by /NH1 complexes is comparable with /1 complexes. While there is 3-fold increase in the apparent Na affinity of /NH1 complexes compared with /1, Na is still required by the /NH1 complexes for measurable levels of ATP hydrolysis. The similar rates of maximum ATP turnover for /NH1 and /1 complexes indicate that the /NH1 complexes are efficient and functional ATPases and that overall enzyme activity has not been severely compromised by the structural change to the subunit. A 3-fold increase in apparent Na affinity for /NH1 in this reaction is similar to the increases in K values estimated for the Na dependence of ouabain binding for these complexes (Fig. 5). Finally, since Na is still required by /NH1 complexes for measurable P release from ATP, despite the ability of these complexes to form a phosphoenzyme from ATP in the absence of Na, it must be concluded that Na is also required at some step in the reaction cycle other than phosphoenzyme formation. The most likely step is in the transition E1P E2P which is thought to accompany release of Na at the extracellular surface of the cell membrane (Fig. 1). Gadsby et al.(48) and Hilgemann (49) have shown that release of Na is voltage-sensitive. The data reported here suggest that both /1 and /NH1 are capable of moving through the reaction cycle to the step where Na would be released, but that the reaction cycle cannot proceed beyond this step when the ion sites are not occupied. In the presence of sodium, the stoichiometry of ion transport by Na/K-ATPase is 3 Na ions transported per ATP molecule hydrolyzed. It is possible that the structural differences between 1, HK, and the chimeric NH1 subunit that result in changes in the apparent affinity for Na are the result of structural perturbations of only one ion binding site or else the cooperative interactions between the binding of multiple Na ions to one or more sites could be affected.

Overall, the chimeric NH1 subunit appears to reduce or eliminate the role of Na in formation of a phosphoenzyme from ATP. It is interesting to note that this effect of the NH1 subunit is not seen with either the 1 or the HK subunits, which are the parent molecules for NH1. Some effect is seen with 3/HK complexes in promoting ATP-dependent ouabain binding at low (1-2 mM) concentrations of Na (Fig. 5). These complexes show only a small amount of ouabain binding in the absence of Na, however, suggesting that Na is still important in increasing the rate of phosphoenzyme formation. Thus, the influence of subunit structure on Na interactions with the pump can not be attributed to one structural region of the subunit, in contrast to the effect of subunit structure on interactions with K(21) . Instead, the results reported here suggest that multiple regions of the subunit interact with multiple regions of the subunit. With the chimeric NH1 subunit, these interactions cause a reduction in the activation energy barrier to phosphoenzyme formation from ATP that is normally reduced by Na. These results provide additional evidence that the subunit is an intimate partner with the subunit in the formation and activity of Na/K-ATPase.

  
Table: Percentage of ouabain binding sites occupied in 3 min

Ouabain binding to yeast membranes containing the indicated subunits was measured after 3 min under the indicated conditions and after 60 min in a Mg- and P-dependent reaction as described under ``Experimental Procedures.'' All reactions were done in the presence of 5 mM MgCl.


  
Table: Sodium-dependent ATP hydrolysis by yeast membranes expressing 1 and 3 subunits with 1 or NH1 subunits

Values for K(Na), the maximum velocity of ATP hydrolysis (µmol/min/mg), and the Hill coefficient (n) were obtained from the experiments shown in Fig. 8. The turnover number was obtained from the ratio V/B, where B is the number of ouabain binding sites determined by equilibrium ouabain binding. Values represent the mean of at least three different experiments done in duplicate.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM28673 (to R. A. F.), American Heart Association Initial Investigator Award 983 F1-2 (to K. A. E.), and American Heart Association Research Fellowship 1014 F1-1 (to R.-M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Dept. of Physiology & Biophysics, USC School of Medicine, 2025 Zonal Ave., Los Angeles, CA 90033.

The abbreviations used are: Na/K-ATPase, sodium- and potassium-transport adenosine triphosphatase (EC 3.6.1.37); 1, the sheep 1 subunit isoform of Na/K-ATPase; 3, the rat 3 subunit isoform of Na/K-ATPase; 1, the rat 1 subunit isoform of Na/K-ATPase; HK, the subunit of rat gastric H/K-ATPase; AMP-PNP, adenosine 5`-(,)-iminodiphosphate.

K. A. Eakle, unpublished data.

K. Wang and R. A. Farley, unpublished result.


ACKNOWLEDGEMENTS

We thank Daun S. Putnam and Ema Hrouda for technical support. We thank Ronald Hitzeman (Genentech) for providing the YEp1PT yeast expression vector and yeast strain 30-4, Edward Benz (Yale) for providing the rat 3 and rat 1 cDNAs, Robert Levenson (Yale) for providing the rat HK cDNA and J. J. H. H. M. de Pont for helpful discussions.


REFERENCES
  1. Forbush, B., III, Kaplan, J. H., and Hoffman, J. F.(1978) Biochemistry 17, 3667-3676 [Medline] [Order article via Infotrieve]
  2. Fallows, D., Kent, R. B., Nelson, D. L., Emanuel, J. R., Levenson, R., and Housman, D. E.(1987) Mol. Cell. Biol. 7, 2985-2987 [Medline] [Order article via Infotrieve]
  3. Price, E., and Lingrel, J. B.(1988) Biochemistry 27, 8400-8408 [Medline] [Order article via Infotrieve]
  4. Noguchi, S., Ohta, T., Takeda, K., Ohtsubo, M., and Kawamura, M.(1988) Biochem. Biophys. Res. Commun. 155, 1237-1243 [Medline] [Order article via Infotrieve]
  5. Canessa, C. M., Horisberger, J.-D., Louvard, D., and Rossier, B. C. (1992) EMBO J. 11, 1681-1687 [Abstract]
  6. Ishii, T., and Takeyasu, K.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8881-8885 [Abstract]
  7. Tran, C. M., Huston, E. E., and Farley, R. A.(1994) J. Biol. Chem. 269, 6558-6565 [Abstract/Free Full Text]
  8. Tran, C. M., Scheiner-Bobis, G., Schoner, W., and Farley, R. A.(1994) Biochemistry 33, 4140-4147 [Medline] [Order article via Infotrieve]
  9. Goldshleger, R., Tal, D. M., Moorman, J., Stein, W. D., and Karlish, S. J. D.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6911-6915 [Abstract]
  10. Arguello, J. M., and Kaplan, J. H.(1994) J. Biol. Chem. 269, 6892-6899 [Abstract/Free Full Text]
  11. Noguchi, S., Mishina, M., Kawamura, M., and Numa, S.(1987) FEBS Lett. 225, 27-32 [CrossRef][Medline] [Order article via Infotrieve]
  12. Horowitz, B., Eakle, K. A., Scheiner-Bobis, G., Randolph, G. R., Chen, C. Y., Hitzeman, R. A., and Farley, R. A..(1990) J. Biol. Chem. 265, 4189-4192 [Abstract/Free Full Text]
  13. Geering, K., Kraehenbuhl, J., and Rossier, B. C.(1987) J. Cell Biol. 105, 2613-2619 [Abstract]
  14. Takeyasu, K., Tamkun, M. M., Renaud, K. J., and Fambrough, D. M. (1988) J. Biol. Chem. 263, 4347-4354 [Abstract/Free Full Text]
  15. Martin-Vassalo, P., Dackowski, W., Emanuel, J. R., and Levenson, R. (1989) J. Biol. Chem. 264, 4613-4618 [Abstract/Free Full Text]
  16. Gloor, S., Antonicek, H., Sweadner, K. J., Pagliusi, S., Rainer, F., Moos, M., and Schachner, M.(1990) J. Cell Biol. 110, 165-174 [Abstract]
  17. Shull, G. E.(1990) J. Biol. Chem. 265, 12123-12126 [Abstract/Free Full Text]
  18. Canfield, V. A., Okamoto, C. T., Chow, D., Dorfman, J., Gros, P., Forte, J. G., and Levenson, R.(1990) J. Biol. Chem. 265, 19878-19884 [Abstract/Free Full Text]
  19. Reuben, M. A., Lasater, L. S., and Sachs, G.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6767-6771 [Abstract]
  20. Eakle, K. A., Kabalin, M. A., Wang, S.-G., and Farley, R. A.(1994) J. Biol. Chem. 269, 6550-6557 [Abstract/Free Full Text]
  21. Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 834-2838
  22. Jaunin, P., Jaisser, F., Beggah, A. T., Takeyasu, K., Mangeat, P., Rossier, B. C., Horisberger, J.-D., and Geering, K.(1993) J. Cell Biol. 123, 1751-1759 [Abstract]
  23. Chow, D. C., Browning, C. M., and Forte, J. G.(1992) Am. J. Physiol. 263, C39-C46
  24. Lutsenko, S., and Kaplan, J. H.(1993) Biochemistry 32, 6737-6743 [Medline] [Order article via Infotrieve]
  25. Forbush, B., III(1983) Curr. Top. Membr. Transp. 19, 167-201
  26. Yoda, S., and Yoda, A.(1987) J. Biol. Chem. 262, 103-109 [Abstract/Free Full Text]
  27. Charnock, J. S., and Post, R. L.(1963) Nature 199, 910-911
  28. Post, R. L., Sen, A. K., and Rosenthal, A. S.(1965) J. Biol. Chem. 240, 1437-1445 [Free Full Text]
  29. Albers, R. W., Koval, G. I., and Siegel, G. I.(1968) Mol. Pharmacol. 4, 325-336
  30. Eakle, K. A., Horowitz, B., Kim, K. S., Levenson, R., and Farley, R. A. (1991) in The Sodium Pump: Recent Developments (Kaplan, J. H., and De Weer, P., eds) pp. 125-129, Rockefeller University Press, NY
  31. Sherman, F., Fink, G. R., and Lawrence, C. W.(1979) Methods in Yeast Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  32. Ito, H., Fukuda, Y., Murata, K., and Kimura, A.(1983) J. Bacteriol. 153, 163-168 [Medline] [Order article via Infotrieve]
  33. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.(1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  34. Jorgensen, P. L.(1974) Biochim. Biophys. Acta 356, 36-52 [Medline] [Order article via Infotrieve]
  35. Cantley, L. C.(1981) Curr. Top. Bioenerg. 11, 201-237
  36. Glynn, I. M., and Karlish, S. J. D(1990) Annu. Rev. Biochem. 59, 171-205 [CrossRef][Medline] [Order article via Infotrieve]
  37. Norby, J. G., Klodos, I., and Christiansen, N. O.(1983) J. Gen. Physiol. 82, 725-759 [Abstract]
  38. Wallick, E. T., and Schwartz, A.(1988) Methods Enzymol. 156, 201-213 [Medline] [Order article via Infotrieve]
  39. Hootman, S. R., and Ernst, S. A.(1988) Methods Enzymol. 156, 213-229 [Medline] [Order article via Infotrieve]
  40. Schuurmans Stekhoven, J. M. A. H., Swarts, H. G. P., de Pont, J. J. H. H. M., and Bonting, S. L.(1985) Biochim. Biophys. Acta 815, 16-24 [Medline] [Order article via Infotrieve]
  41. Schuurmans Stekhoven, J. M. A. H., Swarts, H. G. P., Helmich-de-Jong, M. L., de Pont, J. J. H. H. M., and Bonting, S. L.(1986) Biochim. Biophys. Acta 854, 21-30 [Medline] [Order article via Infotrieve]
  42. Hara, Y., and Nakao, M.(1986) J. Biol. Chem. 261, 12655-12658 [Abstract/Free Full Text]
  43. Polvani, C., and Blostein, R.(1988) J. Biol. Chem. 263, 16757-16763 [Abstract/Free Full Text]
  44. Forbush, B., and Klodos, I.(1991) in The Sodium Pump: Structure, Mechanism, and Regulation (Kaplan, J. H., and De Weer, P., eds) pp. 211-225, Rockefeller University Press, NY
  45. Jewell, E. A., and Lingrel, J. B.(1991) J. Biol. Chem. 266, 16925-16930 [Abstract/Free Full Text]
  46. Horisberger, J.-D., Jaunin, P., Reuben, M. A., Lasater, L. S., Chow, D. C., Forte, J. G., Sachs, G., Rossier, B. C., and Geering, K.(1991) J. Biol. Chem. 266, 19131-19134 [Abstract/Free Full Text]
  47. Jaisser, F., Canessa, C. M., Horisberger, J.-D., and Rossier, B. C. (1992) J. Biol. Chem. 267, 16895-16903 [Abstract/Free Full Text]
  48. Gadsby, D. C., Rakowski, R. F., and DeWeer, P.(1993) Science 260, 100-103 [Medline] [Order article via Infotrieve]
  49. Hilgemann, D. W.(1994) Science 263, 1429-1432 [Medline] [Order article via Infotrieve]

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