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J Biol Chem, Vol. 274, Issue 17, 11604-11610, April 23, 1999

The -Subunits of Na⁺,K⁺-ATPase and Gastric H⁺,K⁺-ATPase Have a High Preference for Their Own -Subunit and Affect the K⁺ Affinity of These Enzymes^*

Jan B. Koenderink, Herman G. P. Swarts, Harm P. H. Hermsen, and Jan Joep H. H. M. De Pont

From the Department of Biochemistry, Institute of Cellular Signaling, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The - and -subunits of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the -subunits resulted in coprecipitation of the accompanying -subunit independent of the type of -subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na⁺,K⁺-ATPase and the -subunit of H⁺,K⁺-ATPase (NaKHK) showed an ATPase activity, which was only 12 ± 4% of the activity of the Na⁺,K⁺-ATPase with its own -subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H⁺,K⁺-ATPase and the -subunit of Na⁺,K⁺-ATPase (HKNaK) showed an ATPase activity which was 9 ± 2% of that of the recombinant H⁺,K⁺-ATPase. In addition, the apparent K⁺ affinity of hybrid NaKHK was decreased, while the apparent K⁺ affinity of the opposite hybrid HKNaK was increased. The hybrid NaKHK could be phosphorylated by ATP to a level of 21 ± 7% of that of Na⁺,K⁺-ATPase. These values, together with the ATPase activity gave turnover numbers for NaK and NaKHK of 8800 ± 310 min¹ and 4800 ± 160 min¹, respectively. Measurements of phosphorylation of the HKNaK and HK enzymes are consistent with a higher turnover of the former. These findings suggest a role of the -subunit in the catalytic turnover. In conclusion, although both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase have a high preference for their own -subunit, they can function with the -subunit of the other enzyme, in which case the K⁺ affinity and turnover number are modified.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase belong to the family of P-type ATPases, which transport ions across the plasma membrane (1). The Na⁺,K⁺-ATPase is found in almost all animal cells and is essential for the maintenance of cellular ion gradients, whereas the gastric H⁺,K⁺-ATPase is located in parietal cells of the gastric mucosa, where it is responsible for acid secretion by the stomach. These X⁺,K⁺-ATPases¹ couple ATP hydrolysis to countertransport of X⁺ (Na⁺ or H⁺) and K⁺ as can be described by the Post-Albers scheme (2-4). Both ATPases consist of an - and a -subunit, which assemble with a 1:1 stoichiometry to form a stable heterodimer. The catalytic ₁-subunits of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase share a high degree of identity (63%), in contrast to their heavily glycosylated ₁-subunits, which are structurally similar but only 30% identical.

Assembly of the - and -subunits is important for conformational stability of the functional holoenzyme (5, 6). This formation of a complex between - and -subunits is also essential for enzyme activity (7-9) and occurs before the subunits are transported from the endoplasmic reticulum to the plasma membrane (10). Lemas et al. (11) showed that the carboxyl-terminal 161 amino acids of the Na⁺,K⁺-ATPase -subunit are sufficient for assembly with the -subunit. More recently, Colonna et al. (12) demonstrated that only four amino acids (SYGQ) in the extracellular loop between the predicted transmembrane helixes 7 and 8 are crucial for - subunit interactions. These four evolutionary conserved amino acids are also present in the -subunit-binding region Arg⁸⁹⁸-Thr⁹²⁸ of the H⁺,K⁺-ATPase -subunit (13). In addition, Wang et al. (14) revealed that the Na⁺,K⁺-ATPase -subunit containing Gln⁹⁰⁵-Val⁹³⁰ of the gastric H⁺,K⁺-ATPase -subunit (including SYGQ) preferentially assembles with the H⁺,K⁺-ATPase -subunit. Many investigators have maintained that the extracellular domain as well as the cytoplasmic and transmembrane domains of the -subunit are important for assembly with the -subunit (13, 15-23).

Eventually the enzymes are transported either to the plasma membrane (Na⁺,K⁺-ATPase) or to the tubulovesicles (H⁺,K⁺-ATPase). Elegant studies by Caplan and co-workers (24, 25) have localized the sorting signal of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase to a sequence of eight amino acids present in the fourth predicted transmembrane domain of the -subunit protein. In the cytoplasmic tail of the H⁺,K⁺-ATPase -subunit a functional endocytosis signal was found. The presence of this motif accounts for the returning of the pump from the apical cell membrane to its intracellular storage compartment resulting in inactivation of acid secretion (26).

Studies of the hybrid ATPase consisting of the Na⁺,K⁺-ATPase -subunit and the H⁺,K⁺-ATPase -subunit have sought to analyze the function of the -subunit. This hybrid ATPase binds ouabain and transports cations across the membrane (27, 28). The extracellular region of the H⁺,K⁺-ATPase -subunit is probably responsible for the slightly higher apparent Na⁺ affinity and the lower apparent K⁺ affinity of this hybrid compared with the Na⁺,K⁺-ATPase (20, 29). In recent years, it has been demonstrated that the predicted transmembrane segments 4, 5, and 6 are involved in cation occlusion (30, 31). The -subunits of both enzymes probably participate in stabilizing this occluded cation intermediate (32, 33). The question arises as to whether the complementary hybrid, consisting of the catalytic subunit of the H⁺,K⁺-ATPase and the -subunit of the Na⁺,K⁺-ATPase, also possesses catalytic activity. We therefore measured both the Na⁺,K⁺-ATPase activity and the H⁺,K⁺-ATPase activity for both hybrid ATPases. Furthermore, this study examines the possible role of -subunits in the apparent K⁺ affinity of both K⁺-dependent ATPases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression Constructs-- The rat gastric H⁺,K⁺-ATPase -subunit cDNA (9) was digested with BglII and ligated into the BamHI site of the pFastbacdual vector (HK) (Life Technologies, Breda, The Netherlands). The -subunit cDNA of the rat gastric H⁺,K⁺-ATPase (9) was digested with BamHI and ligated into the BbsI site of the pFastbacdual vector containing the H⁺,K⁺-ATPase -subunit (HK). The rat Na⁺,K⁺-ATPase ₁-subunit cDNA (34) HindIII fragment was ligated into the HindIII site of the pFastbacdual vector (NaK, with a shortened multiple cloning site through a BamHI-XbaI deletion) or into the pFastbacdual vector (also with BamHI-XbaI deletion) containing the H⁺,K⁺-ATPase -subunit (NaKHK). The sheep Na⁺,K⁺-ATPase ₁-subunit cDNA (35) was digested with SmaI and SpeI and ligated into the SmaI and NheI site of the pFastbacdual vector containing the Na⁺,K⁺-ATPase ₁-subunit (NaK) or into the XhoI and NheI site of the pFastbacdual vector containing the H⁺,K⁺-ATPase -subunit (HKNaK). All -subunits were cloned downstream of the polyhedrin promoter, and all -subunits downstream of the p10 promoter. As mock, the baculovirus DZ1, only expressing -galactosidase, was used (9).

Production of Recombinant Viruses-- Competent DH10bac Escherichia coli cells (Life Technologies, Breda, The Netherlands) harboring the baculovirus genome (bacmid) and a transposition helper vector, were transformed with the pFastbacdual transfer vector containing different cDNAs. Upon transition between the Tn7 sites in the transfer vector and the bacmid, recombinant bacmids were selected and isolated (36). Subsequently, insect Sf9 cells were transfected with recombinant bacmids using Cellfectin reagent (Life Technologies). After 3 days, the recombinant baculoviruses were harvested and used to infect Sf9 cells at a multiplicity of infection of 0.1. Four days after infection, the amplified viruses were harvested.

Preparation of Membranes-- Sf9 cells were grown at 27 °C in 100-ml spinner flask cultures (9). For production of the ATPases subunits 1.0-1.5 × 10⁶ cells/ml were infected at a multiplicity of infection of 1-3 in Xpress medium (BioWittaker, Walkersville, MD) containing 1% ethanol (37) and incubated for 3 days. The Sf9 cells were harvested by centrifugation at 2,000 × g for 5 min, and resuspended at 0 °C in 0.25 M sucrose, 2 mM EDTA, and 25 mM Hepes/Tris (pH 7.0). The membranes were sonicated twice for 30 s at 60 W (Branson Power Co., Denbury, CT), after which the disrupted cells were centrifugated at 10,000 × g for 30 min. The supernatant was recentrifugated at 100,000 × g for 60 min and the pelleted membranes were resuspended in the above mentioned buffer and stored at 20 °C. Native H⁺,K⁺-ATPase and Na⁺,K⁺-ATPase were isolated from the rat gastric mucosa and the sheep kidney outer medulla, respectively (38). These tissues were homogenized and centrifuged at 10,000 × g for 20 min at 4 °C. The supernatant was recentrifuged at 100,000 × g for 30 min. The pelleted membranes were resuspended in the above mentioned buffer and stored at 20 °C.

Protein Determination-- Protein was determined with the modified Lowry method described by Peterson (39) using bovine serum albumin as a standard.

Western Blotting-- Protein samples from the membrane fraction were solubilized in SDS-PAGE sample buffer and separated on SDS gels containing 10% acrylamide according to Laemmli (40). For immunoblotting, the separated proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The - and -subunits of the gastric H⁺,K⁺-ATPase were detected with the polyclonal antibody HKB (41) and the monoclonal antibody 2G11 (42), respectively. The ₁- and ₁-subunits of Na⁺,K⁺-ATPase were detected with the polyclonal antibody L25 (43) and the monoclonal antibody M17-P5-F11 (44), respectively. The primary antibodies were detected using anti-mouse and anti-rabbit secondary antibodies, which were labeled with peroxidase (DAKO A/S, Denmark).

Immunoprecipitation-- Immunoprecipitation was performed essentially as described (45). Either 10 µg of monoclonal antibody 1F11 directed to the H⁺,K⁺-ATPase -subunit (46) or 10 µl of the polyclonal serum L25 directed to the Na⁺,K⁺-ATPase -subunit (43) was added to 20 µl of protein A immobilized on agarose (50% (w/v), KemEnTec, Copenhagen, Denmark) and resuspended in 500 µl of 10 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 0.05% Nonidet P-40 (Fluka, Bornem, Belgium). This mixture was incubated for 1 h at 4 °C with constant agitation, after which the protein A immobilized on agarose was washed twice in the above mentioned buffer and once in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Nonidet P-40. Crude membrane proteins (~700 µg) were solubilized in 500 µl of buffer containing 1% (w/v) octaethylene glycol monododecylether (C₁₂E₈, Sigma), 10 mM Tris-HCl (pH 8.0), and 150 mM NaCl for 1 h at 4 °C. Following centrifugation at 10,000 × g for 5 min, the supernatant was incubated with the antibodies bound to the protein A immobilized on agarose for 1 h at 4 °C. The immunoprecipitates were collected by centrifugation for a few seconds at 10,000 × g, washed 3 times in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and solubilized in SDS-PAGE sample buffer. After SDS-PAGE the ATPases subunits in the precipitates were identified by immunoblotting, using the biotin-labeled antibodies L25 (43), M17-P5-F11 (44), and 2G11 (42). The antibody HK9 was used for detection of the H⁺,K⁺-ATPase -subunit (47). The primary antibodies labeled with biotin were detected using streptavidin labeled with peroxidase (Jackson ImmunoResearch, West Grove, PA), while HK9 was detected with anti-rabbit secondary antibody, which was also labeled with peroxidase (DAKO A/S, Denmark).

ATPase Activity Assay-- The ATPase activity was determined with a radiochemical method (48). For this purpose 0.6-7 µg of Sf9 membranes were added to 100 µl of medium, which contained 1 mM MgCl₂, 0.2 mM EGTA, 0.1 mM EDTA, 1 mM NaN₃, and 25 mM Tris-HCl (pH 7.0). For determination of Na⁺,K⁺-ATPase activity, 100 µM [-³²P]ATP, 100 mM NaCl, and 0.01 mM ouabain (in order to inhibit endogenous Na⁺,K⁺-ATPase from Sf9 cells) were present. The specific activity is presented as the difference in activity with and without 10 mM KCl. In the incubation medium used for the measurements of the K⁺ activation curve only 10 mM NaCl was present. For determination of H⁺,K⁺-ATPase activity, 10 µM [-³²P]ATP (specific activity 100-500 mCi mmol¹), 1 mM KCl, and 0.1 mM ouabain were present. The specific activity is presented as the difference in activity with and without 0.1 mM SCH 28080. After incubation at 37 °C the reaction was stopped by adding 500 µl of 10% (w/v) charcoal in 6% (w/v) trichloroacetic acid and after incubation at 0 °C the mixture was centrifuged for 30 s (10,000 × g). To 200 µl of the clear supernatant, containing the liberated inorganic phosphate (³²P_i), 4 ml of OptiFluor (Canberra Packard, Tilburg, The Netherlands) was added and the mixture was analyzed by liquid scintillation analysis. Blanks were prepared by incubating in the absence of enzyme.

ATP Phosphorylation Capacity-- ATP phosphorylation was determined as described (37). Sf9 membranes (6-70 µg) were incubated at 0 °C in 25 mM Tris acetic acid (pH 6.0), 1 mM MgCl₂ in a volume of 50 µl. For phosphorylation of Na⁺,K⁺-ATPase, 100 mM NaCl was added to the incubation buffer. This phosphorylation is presented as the difference with and without 10 mM KCl. Phosphorylation of H⁺,K⁺-ATPase is presented as the difference with and without 0.1 mM SCH 28080. After 30-60 min preincubation, 10 µl of 0.6 µM [-³²P]ATP was added and incubated for 10 s at 0 °C. The reaction was stopped by adding 5% trichloroacetic acid in 0.1 M phosphoric acid and the phosphorylated protein was collected by filtration over a 0.8-µm membrane filter (Schleicher and Schuell, Dassel, Germany). After repeated washing the filters were analyzed by liquid scintillation analysis. Phosphorylation at different ATP concentrations was performed at 21 °C and for Na⁺,K⁺-ATPase in the presence of 20 µg/ml oligomycin (a mixture of A, B, and C; Sigma).

Calculations-- The K_0.5 value is defined as the concentration of effector (K⁺) giving the half-maximal activation and the IC₅₀ as the value giving 50% inhibition of the maximal activation. Data are presented as mean values with standard error of the mean. Differences were tested for significance by means of the Student's t test.

Materials-- The cDNA clones of the H⁺,K⁺-ATPase - and -subunits and the rat and sheep cDNA clones of the Na⁺,K⁺-ATPase ₁- and ₁-subunits were provided by Drs. G. E. Shull and J. B. Lingrel, respectively. Cellfectin, competent DH10bac E. coli cells, and all enzymes used for DNA cloning were purchased from Life Technologies Inc. [-³²P]ATP (3000 Ci mmol¹) was purchased from Amersham (Buckinghamshire, United Kingdom). SCH 28080, kindly provided by Dr. A. Barnett, Schering-Plow, Kenilworth, NJ, was dissolved in ethanol and diluted to its final concentration of 0.1 mM in 0.2% ethanol. The antibodies 2G11 and M17-P5-F11 were gifts of Drs. J. Forte (Berkeley) and W. J. Ball Jr. (Cincinnati), respectively. Dr. J. V. Møller (Aarhus, Denmark) provided antibody L25 and Dr. M. Caplan (Yale) antibodies HKB and HK9.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recombinant baculoviruses expressing Na⁺,K⁺-ATPase, H⁺,K⁺-ATPase, their -subunits, and their hybrids were produced and Sf9 cells were infected. The membrane fractions of these Sf9 cells expressing the recombinant proteins were isolated and Western blot analysis revealed comparable expression patterns (Fig. 1). Both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase -subunits detected with antibodies L25 (43) and HKB (41), respectively, had an apparent molecular mass of about 100 kDa. The antibody M17-P5-F11 (44) visualized both a carbohydrate-free and a core-glycosylated form of the Na⁺,K⁺-ATPase -subunit. The monoclonal antibody 2G11 (42) also recognized a carbohydrate-free and a core-glycosylated form of the H⁺,K⁺-ATPase -subunit. This carbohydrate-free form was of a similar molecular mass as the carbohydrate-free Na⁺,K⁺-ATPase -subunit, but the core-glycosylated Na⁺,K⁺-ATPase -subunit had a lower apparent molecular mass, due to the presence of three glycosylation sites in this subunit in contrast to the six glycosylation sites present in the H⁺,K⁺-ATPase -subunit.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Western blot of Na+,K+-ATPase, H+,K+-ATPase, and their hybrids. Membranes (~10 µg) isolated from infected Sf9 cells were blotted and the presence of Na+,K+-ATPase - and -subunits was detected with antibodies L25 and M17-P5-F11, respectively. The - and -subunits of H+,K+-ATPase were detected with antibodies HKB and 2G11, respectively. The subunits to which the different primary antibodies were directed are indicated on the left and the molecular weights are indicated on the right.

Interaction between the - and -subunits in both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase is essential for a functionally active enzyme. To examine the interaction between - and -subunits in hybrid ATPases an immunoprecipitation assay was performed. The -subunits of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase were immunoprecipitated with antibodies L25 (43) and 1F11 (46), respectively. In control experiments both the Na⁺,K⁺-ATPase isolated from sheep kidney and recombinant Na⁺,K⁺-ATPase were precipitated with antibody L25. Similarly, rat gastric H⁺,K⁺-ATPase and recombinant H⁺,K⁺-ATPase were precipitated by antibody 1F11. With the native enzymes only glycosylated -subunits were precipitated, with apparent molecular masses significantly higher than those of their recombinant counterparts. This is due to the absence of complex glycosylation in Sf9 cells. When the same method was used for the hybrid ATPases, both -subunits were also coprecipitated with the other -subunit (Fig. 2). This indicates that there is not only an interaction between the - and -subunits of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase, but also between the - and -subunits of the two hybrid ATPases. Although quantification in these experiments is rather difficult, there seemed to be less coprecipitated -subunits in the hybrid ATPases than in the wild type ATPases (NaK, HK).

View larger version (60K): [in this window] [in a new window]   Fig. 2.   Western blot of immunoprecipitated - and -subunits. The -subunits of Na+,K+-ATPase and H+,K+-ATPase were bound to antibodies L25 and 1F11, respectively, which complexes were immunoprecipitated with protein A immobilized on agarose. After SDS-PAGE and Western blotting, the -subunits were stained with L25 and HK9. The presence of the possibly coprecipitated Na+,K+-ATPase and H+,K+-ATPase -subunits was detected with M17-P5-F11 and 2G11, respectively. NaK kidney and HK stomach were the isolated enzymes from the kidney and the gastric mucosa, respectively.

When HKNaK was present during immunoprecipitation with antibody L25 (directed against the -subunit of Na⁺,K⁺-ATPase), the -subunit of Na⁺,K⁺-ATPase was unexpectedly precipitated. Also when the H⁺,K⁺-ATPase -subunit was absent the Na⁺,K⁺-ATPase -subunit was precipitated (data not shown). In the absence of antibody L25 the Na⁺,K⁺-ATPase -subunit was not precipitated. These findings suggest that the expressed Na⁺,K⁺-ATPase -subunit assembles with the endogenous Na⁺,K⁺-ATPase -subunit. Antibody L25 apparently recognizes this endogenous -subunit during immunoprecipitation, but not on a Western blot. The absence of unglycosylated -subunit indicates that the assembly with the endogenous -subunit only occurred during the early stage of infection, when the glycosylation machinery is still functional.

Na⁺,K⁺-ATPase activity was measured in the presence of 100 µM ATP and optimal concentrations of Na⁺ (100 mM) and K⁺ (10 mM). Because of the endogenous Na⁺,K⁺-ATPase activity (50-100 nmol mg¹ protein h¹) present in the Sf9 membrane preparations, we determined the ouabain sensitivity for the endogenously present Na⁺,K⁺-ATPase in mock infected cells. The endogenous Na⁺,K⁺-ATPase activity was completely inhibited at 1 × 10⁵ M ouabain, while the recombinant activity was hardly inhibited at this concentration. These findings are in line with those of Lui and Guidotti (49). Using 1 × 10⁵ M ouabain in the assay, we measured the recombinant ATPase activity as the difference between the activity with and without 10 mM K⁺ (Fig. 3A). The activity of NaKHK was 12 ± 4% (n = 3) of the activity of NaK. NaK and mock infected cells did not show Na⁺,K⁺-ATPase activity in the presence of 10⁵ M ouabain.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   ATPase activity in Sf9 membranes infected with recombinant baculoviruses. Membranes obtained from Sf9 cells, expressing Na+,K+-ATPase, H+,K+-ATPase, and their hybrids, were isolated and incubated at 37 °C and pH 7.0 with 10 µM ATP and 1 mM KCl for H+,K+-ATPase activity, while Na+,K+-ATPase activity was determined in the presence of 100 µM ATP, 100 mM NaCl, and 10 mM KCl. The Na+,K+-ATPase activity (A) and the H+,K+-ATPase activity (B) were measured as described under "Experimental Procedures." The values presented are the mean ± S.E. of three experiments. *, significantly different from the mock and -subunit ATPase activity (p < 0.05).

In order to measure K⁺-stimulated H⁺,K⁺-ATPase activity we used a suboptimal (10 µM) ATP concentration since at higher ATP concentrations the endogenous activity increases relatively more than the H⁺,K⁺-ATPase activity. The K⁺ concentration (1 mM) used was optimal for H⁺,K⁺-ATPase activity under these conditions (50). The hybrid HKNaK showed an SCH 28080-sensitive ATPase activity which was 7 ± 1% (n = 3) of the HK activity (Fig. 3B). HK and mock did not show any SCH 28080-sensitive ATPase activity. In conclusion, these hybrids possess only a fraction of the ATPase activity of Na⁺,K⁺-ATPase or H⁺,K⁺-ATPase when measured under similar conditions.

To determine if this low ATPase activity of the hybrid ATPases could be due to changed ATP affinities, we measured the ATPase activity at different ATP concentrations (0.03-1 mM). The results are given in a Woolf-Augustinsson-Hofstee plot (Fig. 4). The intercept with the y axis is equal to the maximal ATPase activity at infinite ATP concentrations. The maximal NaK ATPase activity was 1.8 ± 0.6 µmol mg¹ protein h¹, whereas the activity for NaKHK was 0.21 ± 0.03 µmol mg¹ protein h¹ (which was 12 ± 4% of that of NaK, n = 3). The maximal HK ATPase activity was 1.4 ± 0.1 µmol mg¹ protein h¹, whereas the activity for HKNaK was 0.12 ± 0.02 µmol mg¹ protein h¹ (which was 9 ± 2% of that of HK, n = 3). The slope of this graph represents the apparent ATP affinity. The apparent ATP affinities for NaK (197 ± 27 µM, n = 3) and NaKHK (206 ± 5 µM, n = 3) are similar. Also the apparent ATP affinities for HK (17 ± 0.7 µM, n = 3) and HKNaK (13 ± 1.3 µM, n = 3) are not significantly different. This indicates that the lower ATPase activity of the hybrids is not due to a change in ATP affinity.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Woolf-Augustinsson-Hofstee plot of the ATPase activity versus the ATP concentration. Membranes obtained from Sf9 cells, expressing Na+,K+-ATPase (A, ), NaKHK (B, ), H+,K+-ATPase (A, ), or HKNaK (B, ), were isolated and incubated at 37 °C and pH 7.0 with 1 mM KCl for H+,K+-ATPase activity, while Na+,K+-ATPase activity was determined in the presence of 100 mM NaCl and 10 mM KCl. The ATPase activity was measured as described under "Experimental Procedures" using varying concentrations of ATP (0.03-1 mM). The activities obtained with NaK and NaKHK were measured as the difference in activity in the presence of 0.01 and 1 mM ouabain.

Both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase occlude K⁺ and transport this ion across the plasma membrane. We compared the K⁺ dependence of the overall ATPase activity of Na⁺,K⁺-ATPase, H⁺,K⁺-ATPase, and their hybrids. All ATPases showed a biphasic activation curve, in which the maximal ATPase activity was set at 100% (Fig. 5). The increasing part of this curve is due to K⁺ activation of the dephosphorylation step. The decreasing part is due to the competition of K⁺ with H⁺ or Na⁺ at the cytoplasmic activation sites. This directs the enzyme from the E₁ toward the E₂ conformation, which cannot be overcome by the low ATP concentration used. The K_0.5 for K⁺ of NaK was 0.5 ± 0.2 mM and the IC₅₀ was 50 ± 9 mM (Fig. 5A). The curve of the hybrid NaKHK was shifted to the right compared with the curve of NaK. The K_0.5 and the IC₅₀ values of this hybrid were slightly increased to 0.7 ± 0.3 mM (K_0.5) and 103 ± 24 mM (IC₅₀), respectively, when compared with NaK. In contrast, the K⁺ activation curve of HKNaK was shifted to the left compared with the curve of HK (Fig. 5B). Moreover, the K_0.5 for HK (0.07 ± 0.01 mM) was decreased for the hybrid HKNaK (0.02 ± 0.004 mM) and also the IC₅₀ was decreased from 10 ± 0.2 to 3.5 ± 0.7 mM. These shifts in K_0.5 and IC₅₀ values were significant (p < 0.05, n = 3). Thus NaKHK had a slightly decreased K⁺ affinity compared with NaK, while the opposite hybrid HKNaK had an increased K⁺ affinity compared with HK. These findings indicate that the -subunits of both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase influence the K⁺ sensitivity of these enzymes.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of K+ on the ATPase activity in Sf9 membranes infected with recombinant baculoviruses. Membranes obtained from Sf9 cells, expressing Na+,K+-ATPase (), NaKHK (), H+,K+-ATPase (), or HKNaK () were isolated and incubated at 37 °C and pH 7.0 with 10 µM ATP for H+,K+-ATPase activity, while Na+,K+-ATPase activity was determined in the presence of 100 µM ATP and 10 mM NaCl. The K+ activated ATPase activity was measured as described under "Experimental Procedures" using varying concentrations of K+. The specific Na+,K+-ATPase activity is presented as the activity in the presence of 105 M ouabain, minus the ATPase activity of mock infected membranes (A). The specific H+,K+-ATPase activity is presented as the difference in activity with and without SCH 28080 (B). The maximal ATPase activity for each preparation was set at 100% and the values presented are the mean ± S.E. of three experiments. The K0.5 as well as the IC50 values were significantly different between HK and HKNaK (p < 0.05).

The formation of an acid-stable phosphorylated intermediate during the catalytic cycle is a characteristic property of the P-type ATPases. Phosphorylation of Na⁺,K⁺-ATPase was measured with Na⁺ present in the preincubation in order to shift the equilibrium of the enzyme forms toward Na·E₁. NaKHK was phosphorylated for 28 ± 11% compared with NaK (n = 3, Fig. 6A). Unlike phosphorylation of NaKHK, phosphorylation of HKNaK was not visible, whereas HK was normally phosphorylated (Fig. 6B). Changing the temperature to 21 °C (31), longer incubation periods, inhibition with K⁺ instead of SCH 28080, higher ATP concentrations, or addition of triallylamine (51) still did not result in any measurable amount of phosphorylated intermediate. In order to determine the maximal phosphorylation level we measured the phosphorylation level at different ATP concentrations (0.006-0.2 µM) at 21 °C. The data are plotted as the phosphorylation level versus the ATP concentration (Fig. 7A) and the same data are also visualized in a Woolf-Augustinsson-Hofstee plot (Fig. 7B). In the last plot the apparent ATP affinity and the maximal phosphorylation level can be determined more easily. The ATP affinity is equal to the slope of the graph, while the intercept with the y axis is equal to the maximal phosphorylation level. For this interpretation it must be assumed that the distribution of EP forms does not change over the range of concentrations of ATP used. For HK a maximal phosphorylation level of 6.3 ± 0.9 pmol mg¹ protein with an apparent ATP affinity of 23 ± 3 nM (n = 3) was measured. In the reaction mixture where the Na⁺,K⁺-ATPase -subunit was present oligomycin was included, which increased the phosphorylation level by about 30%. The apparent ATP affinities for NaK (12 ± 2 nM, n = 3) and NaKHK (12 ± 1 nM, n = 3) are similar. However, the maximal phosphorylation level for NaK was 3.3 ± 1.0 pmol mg¹ protein, whereas the maximal phosphorylation level for NaKHK was 0.71 ± 0.10 pmol mg¹ protein (which was 21 ± 7% of that of NaK, n = 3). These values, together with the maximal ATPase activity (determined at infinite ATP concentrations) give turnover numbers for NaK and NaKHK of 8800 ± 310 min¹ and 4800 ± 160 min¹, respectively. These data are not corrected for the suboptimal K⁺ concentration for the hybrid ATPase in the ATPase reaction, which probably will increase the turnover number by about 15%.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   ATP-phosphorylation level in Sf9 membrane preparations infected with recombinant baculovirus. Membranes obtained from Sf9 cells, expressing Na+,K+-ATPase, H+,K+-ATPase, and their hybrids, were isolated and incubated for 10 s at 0 °C and pH 6.0 in the presence of 0.1 µM ATP for H+,K+-ATPase, while for Na+,K+-ATPase also 100 mM NaCl was present. The Na+,K+-ATPase phosphorylation level (A) and the H+,K+-ATPase phosphorylation level (B) were measured as described under "Experimental Procedures." The values presented are the mean ± S.E. of three experiments. *, significantly different from the mock and -subunit phosphorylation levels (p < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   ATP dependend phosphorylation level. Membranes obtained from Sf9 cells, expressing Na+,K+-ATPase (), NaKHK (), or H+,K+-ATPase (), were isolated and incubated for 10 s at 21 °C and pH 6.0 for H+,K+-ATPase, while for Na+,K+-ATPase 100 mM NaCl was also present. The phosphorylation level was measured as described under "Experimental Procedures" varying the concentrations of ATP (0.006-0.2 µM). The phosphorylation levels obtained with NaK and NaKHK were corrected for that of NaK. A, phosphorylation level as a function of the ATP concentration. B, data from A represented as a Woolf-Augustinsson-Hofstee plot.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The -subunits of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase are involved in correct folding (10) of the -subunits. In addition, the -subunit of H⁺,K⁺-ATPase plays a role in endocytosis of the enzyme (24). Most probably, both -subunits are also involved in the modulation of the enzyme activity: Na⁺ and K⁺ affinities of the Na⁺,K⁺-ATPase were changed when the Na⁺,K⁺-ATPase -subunit was replaced by the H⁺,K⁺-ATPase -subunit (20, 29). In the present study we demonstrate that the hybrid consisting of the H⁺,K⁺-ATPase -subunit and the Na⁺,K⁺-ATPase ₁-subunit has a low ATPase activity with a high apparent K⁺ affinity and probably a high turnover number as compared with H⁺,K⁺-ATPase.

Recombinant Na⁺,K⁺-ATPase, H⁺,K⁺-ATPase, and their hybrids were produced with the baculovirus expression system. The molecular mass of all -subunits expressed was similar to that of the isolated -subunits. However, the recombinant -subunits were less glycosylated than the isolated -subunits, as has been reported before (9, 52). Expression levels of the subunits of each hybrid ATPase were comparable to those of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase, respectively.

Assembly of - and -subunits is a crucial step in the formation of active X⁺,K⁺-ATPases (7-9). The minimal -subunit-binding site (12) in the -subunits of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase are identical and therefore an interaction between the - and -subunits of the different ATPases seems likely. Both -subunits are only 30% identical, but have a high structural similarity. In the hybrid ATPase NaKHK the subunits cross-assembled in a co-immunoprecipitation experiment. Others also observed an interaction between the Na⁺,K⁺-ATPase -subunit and the H⁺,K⁺-ATPase -subunit (27, 28, 53). A possible interaction between the H⁺,K⁺-ATPase -subunit and the Na⁺,K⁺-ATPase -subunit has not been demonstrated so far. When in our experiments the -subunit of the hybrid ATPase HKNaK was precipitated by anti-Na⁺,K⁺-ATPase antibodies, the -subunit of Na⁺,K⁺-ATPase coprecipitated. However, for both hybrid ATPases there seemed to be less coprecipitated -subunits compared with the wild type enzymes, indicating a less efficient assembly between the different subunits of the hybrid ATPases. This could be due to the 70% difference in amino acid composition or to the difference in glycosylation between the two -subunits.

Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase need hydrolysis of ATP to transport cations across the membrane. Accordingly, functional hybrid ATPases should also have ATPase activity. We determined that the hybrid ATPase NaKHK possessed a low ATPase activity which was about 12% of the activity of recombinant Na⁺,K⁺-ATPase. Hybrid ATPase activity has not been measured so far, although in yeast expressing the Na⁺,K⁺-ATPase ₁-subunit and the H⁺,K⁺-ATPase -subunit ouabain binding was observed (27). ATPase activity has been measured in membranes isolated from yeast expressing the Na⁺,K⁺-ATPase ₁-subunit and a chimeric ₁-subunit (29). In oocytes expressing NaKHK a small Na⁺,K⁺-pump current and Rb⁺ uptake has been detected (28), although this could not be confirmed by Ueno et al. (54), which might be due to the very low activity of this hybrid ATPase. None of these assays were performed for the hybrid ATPase HKNaK. When we measured the SCH 28080-sensitive ATPase activity, this hybrid exhibited also a low ATPase activity, which was only 9% of the activity of recombinant H⁺,K⁺-ATPase. Both hybrid ATPases have ATP hydrolyzing activity, although this activity is much lower than that of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase. The ATP affinities of the hybrids are similar to those of the wild type ATPases. So, the lower ATPase activity of hybrid ATPases might result from a decreased amount of functional hybrid ATPase molecules.

X⁺,K⁺-ATPases need K⁺ occlusion before they can dephosphorylate (31) and probably the -subunit is involved in this K⁺ occlusion (32, 33). When the Na⁺,K⁺-ATPase -subunit was replaced by the H⁺,K⁺-ATPase -subunit the K⁺ affinity in the ATPase reaction decreased similarly as has been reported in ouabain binding experiments by Eakle et al. (20). However, the K⁺ affinity of the complementary hybrid ATPase HKNaK was increased as compared with that of H⁺,K⁺-ATPase. Thus the apparent K⁺ affinities of Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase are partly modulated through their -subunits. In summary, the -subunit of Na⁺,K⁺-ATPase, as compared with the -subunit of H⁺,K⁺-ATPase, gives the enzyme a higher apparent K⁺ affinity.

When K⁺ ions are absent during incubation, the enzymes are presumably accumulated in the phosphorylated state. The hybrid ATPase NaKHK was phosphorylated to 21% of the Na⁺,K⁺-ATPase phosphorylation level, which is slightly higher than the percentage activity obtained in the ATPase reaction. This lower ATPase activity and phosphorylation level of the hybrid must mainly be caused by a less efficient subunit assembly. Surprisingly, with the hybrid HKNaK we were not able to measure any specific phosphorylation. Although no K⁺ is added to the reaction mixture it still contained about 5 µM K⁺ as determined by flame photometry. The decrease in K_0.5 directs the enzyme from the E₂-P into the E₂ conformation, while the decrease in IC₅₀ directs the enzyme from the E₁ into the E₂ conformation. Both these processes inhibit accumulation of hybrid HKNaK in the E₂-P conformation and drive the enzyme into the E₂ conformation. It is unlikely, however, that the low amount of K⁺ present accounts for the total absence of any phosphorylated intermediate.

Another explanation for the absence of a phosphorylated intermediate in the hybrid HKNaK is an increased turnover number. The H⁺,K⁺-ATPase -subunit decreases the turnover number of the hybrid NaKHK as compared with that of NaK significantly. The assumption that the Na⁺,K⁺-ATPase -subunit increases the turnover number of the hybrid HKNaK, as compared with HK, seems likely. This higher turnover number would then be responsible for a lower phosphorylation level for HKNaK. If the increase in turnover number is of the same magnitude as the 1.8-fold decrease in the turnover number of the hybrid NaKHK as compared with NaK, then the maximal EP level of HKNaK is 0.3 pmol mg¹ protein, which is below the detection limit.

The suggestion of a relationship between the K⁺ affinity and the turnover of the ATPases, which both are influenced by the -subunit, is tempting. The deocclusion step for Na⁺,K⁺-ATPase is the rate-limiting step, while for the H⁺,K⁺-ATPase the dephosphorylation step (this is the occlusion of K⁺) is rate-limiting. This rate-limiting step must be accelerated if the turnover number is raised. The hybrid HKNaK then not only has an increased K⁺ affinity but also a higher rate of K⁺ stimulated dephosphorylation as compared with HK. Thus, HKNaK occludes K⁺ faster and with a higher affinity than HK, which directly increases the turnover number. The opposite is true for the other hybrid NaKHK, although in this case the K⁺ occlusion becomes rate-limiting.

The findings reported here show that both Na⁺,K⁺-ATPase and H⁺,K⁺-ATPase require their own -subunits for optimal activity. Probably the subunit assembly in the hybrid ATPases is less efficient than in the wild type ATPases. When the -subunits are exchanged, the enzyme activity decreases and the apparent K⁺ affinity of both hybrid ATPases is modified. The Na⁺,K⁺-ATPase -subunit gives the enzyme a higher K⁺ affinity and probably a higher turnover number than the H⁺,K⁺-ATPase -subunit.

	ACKNOWLEDGEMENTS

We thank Dr. A. Barnett for providing SCH 28080 and Drs. M. Caplan, J. Forte, W. J. Ball Jr., and J. V. Møller for generously providing the various antibodies. We also thank Drs. G. E. Shull and J. B. Lingrel for providing the rat cDNA clones of the H⁺,K⁺-ATPase - and -subunits and the rat and sheep cDNA clones of the Na⁺,K⁺-ATPase ₁- and ₁-subunits, respectively.

	FOOTNOTES

^* This work was supported by Netherlands Foundation for Scientific Research (NWO-ALW) Grant 805-05.041.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 31-24-3614260; Fax: 31-24-3540525; E-mail: J.dePont{at}bioch.kun.nl.

	ABBREVIATIONS

The abbreviations used are: X⁺, Na⁺ or H⁺; HK, H⁺,K⁺-ATPase -subunit; NaK, Na⁺,K⁺-ATPase -subunit; NaK, Na⁺,K⁺-ATPase; NaKHK, the hybrid consisting of the Na⁺,K⁺-ATPase -subunit and the H⁺,K⁺-ATPase -subunit; HK, H⁺,K⁺-ATPase; HKNaK, the hybrid consisting of the H⁺,K⁺-ATPase -subunit and the Na⁺,K⁺-ATPase -subunit; Sf, Spodoptera frugiperda; PAGE, polyacrylamide gel electrophoresis; C₁₂E₈, octaethylene glycol monododecylether; SCH 28080, 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine.

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				L. A. Dunbar and M. J. Caplan Ion Pumps in Polarized Cells: Sorting and Regulation of the Na+,K+- and H+,K+-ATPases J. Biol. Chem., August 3, 2001; 276(32): 29617 - 29620. [Abstract] [Full Text] [PDF]

				J. B. Koenderink, H. P. H. Hermsen, H. G. P. Swarts, P. H. G. M. Willems, and J. J. H. H. M. De Pont High-affinity ouabain binding by a chimeric gastric H+,K+-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the alpha 1-subunit of rat Na+,K+-ATPase PNAS, October 10, 2000; 97(21): 11209 - 11214. [Abstract] [Full Text] [PDF]

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