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Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Douglas R. Yingst1, Shang-You Yang2, and Rick Schiebinger2

Departments of 1 Physiology and 2 Internal Medicine, Division of Endocrinology, Wayne State University School of Medicine, Detroit, Michigan 48201

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from ~2 to 15 µmol Pi · mg-1 · min-1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the alpha -subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.

phosphorylation; kinase; phosphatase; sodium; potassium; rat adrenal glomerulosa; rat kidney; dog kidney; affinity chromatography

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

OVER THE LAST 25 years, there have been a number of attempts to develop ouabain-affinity columns. These columns are an attractive idea for the purification and study of Na+-K+-ATPase, because ouabain specifically binds Na+-K+-ATPase. A number of such columns have been made using different procedures (27, 30, 32, 35), but for a variety of reasons, none has proven effective in isolating and purifying an active form of Na+-K+-ATPase. Likewise, none of these columns has subsequently been used for other purposes, perhaps because of the lack of adequate documentation of how they interact with Na+-K+-ATPase.

In this paper we describe a new ouabain-affinity column and demonstrate that it works by two criteria. First, we show that the column specifically binds and releases Na+-K+-ATPase as a function of Na, K, Mg, and ATP. These ligands control how Na+-K+-ATPase attaches to the column in the same manner that they regulate how Na+-K+-ATPase binds to untethered ouabain. For instance, Na+-K+-ATPase binds to ouabain in the presence of Na + ATP + Mg (28) and releases ouabain in the presence of Na + ATP (19). These studies demonstrate that the column interacts with Na+-K+-ATPase as one would predict based on the interactions of Na+-K+-ATPase with untethered ouabain. This principle can be used by others to develop their own uses of the column. Second, we show that the column can be used to purify Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The present purification procedures have been optimized to study the rat alpha 1-isoform of Na+-K+-ATPase, which has a low affinity for ouabain. The development of related procedures should make the column equally useful for studying other forms of Na+-K+-ATPase.

Because of its high affinity and relatively low capacity, we suggest that the column will be particularly useful in purifying Na+-K+-ATPase from cells and tissues that contain modest amounts of this enzyme. For this reason, use of the column will compliment traditional purification methods designed to purify Na+-K+-ATPase from the few tissues that contain abundant Na+-K+-ATPase. The ability to purify Na+-K+-ATPase from cells with more modest levels of Na+-K+-ATPase will help to evaluate the extent to which posttranslational modifications exist and help determine the role they play in regulating Na+-K+-ATPase, which is a major unanswered question in cell physiology (2, 3, 6; for reviews, see Refs. 10, 26).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

ImmunoPure epoxy-activated agarose was obtained from Pierce Chemical (Rockford, IL). Ouabain, CHAPS, leupeptin, Na2ATP, phenylmethylsulfonyl fluoride (PMSF), and phosphatidylcholine were obtained from Sigma Chemical (St. Louis, MO), and SDS was from GIBCO Life Technologies (Gaithersburg, MD). Purified dog kidney Na+-K+-ATPase with a specific activity of ~15 µmol · mg-1 · min-1 was a generous gift of Dr. Svetlana Lutsenko of Oregon Health Science University. This enzyme had been purified by the method of Jorgensen (20) and was stored at -20°C in 20 mM imidazole, 1 mM EDTA, and 20% glycerol, pH 7.5. All chemicals were analytical grade. Anti-rat Na+-K+-ATPase alpha 1-, alpha 2-, and alpha 3-polyclonal antisera were purchased from Upstate Biotechnology (Lake Placid, NY).

Cell and Tissue Preparation

Rat kidney. Rat kidney microsomes were prepared by the procedure of Jorgensen (20). The kidneys were removed from the rat, minced whole on ice, and homogenized with a glass-Teflon Wheaton Potter-Elvehjem homogenizer (1,000 rev/min) in 10 vol of buffer containing 50 mM imidazole (pH 7.4), 2 mM EDTA, and 250 mM sucrose. The homogenate was centrifuged at 6,000 g for 20 min, and the supernatant was spun again at 48,000 g for 60 min at 4°C. The subsequent pellet was suspended in the homogenization buffer at a final protein concentration of ~5 mg/ml and was stored at -70°C.

Rat adrenal cells. Adrenal glomerulosa cells were prepared by collagenase dispersion and maintained as previously described (8). Experiments were performed with modified medium 199 (GIBCO) that contained modified Earle's salts, including 4 mM KCl, 140 mM NaCl, and 20 mM HEPES in place of bicarbonate, pH 7.4.

Preparation of Ouabain-Affinity Matrix

One gram of epoxy-activated agarose containing a 13-atom spacer (catalog no. 20241, Pierce Chemical) was added to 15 ml of a solution containing 10 mM ouabain and 100 mM sodium carbonate buffer (pH 8.5) at room temperature following the instructions from Pierce Chemical. The solution was incubated at 37°C for 20 h with gentle shaking. The swollen agarose (~2 ml) was then washed with 10 ml of the carbonate buffer followed by distilled water. Coupling sites that were still active were blocked by incubation of the agarose with 1 M ethanolamine for 4 h at 37°C, pH 8.5. The agarose was then washed with 200 mM Tris at pH 8.5. Finally, the coupled agarose was washed and stored in 25 mM imidazole and 0.05% NaN3 at 4°C, pH 7.4.

Running the Column Using Previously Purified Dog Na+-K+-ATPase

A column (0.8 × 4 cm) with a removable cap containing 1-2 ml of swollen ouabain-linked agarose was washed at room temperature (20-22°C) with 50 bed volumes of a solution containing 50 mM imidazole, pH 7.4, and then with 5 bed volumes of loading buffer (Table 1) at a flow rate of 0.5-1 ml/min. At the end of the washes, the buffer was drained down to the surface of the agarose, and flow through the column was halted. Previously purified dog kidney Na+-K+-ATPase (6-10 µg/ml bed vol) suspended in 1.5 bed volumes of one of the loading buffers (Table 1) was put on top of the agarose, and the top of the column was closed. The temperature was reduced to 4°C, and the column was rotated end to end with a nutator for 45-60 min. The column was mounted in a vertical position, the cover was removed, the agarose was allowed to settle, and the column was drained from the bottom by gravity until there was no buffer left standing over the agarose (pass-through fraction). The column was then washed by gravity with 10 bed volumes of loading buffer at 4°C. The last 1.5 bed volumes of this wash were collected and saved (last wash, 4°C). The flow through the column was halted, 2 bed volumes of loading buffer were added to the top of the column, and the temperature was raised to 37°C. After 5 min, gravity flow through the column was restarted and the column was washed further with 10 bed volumes of the loading buffer. The last 1.5 bed volumes were collected (last wash, 37o C), leaving no buffer standing over the ouabain-linked agarose. Although not done in the experiments reported here, it is best if 1 bed volume of elution buffer now be added to the top of the column and collected at the bottom. This step is to remove ligands in the wash buffer that promote ouabain binding to Na+-K+-ATPase. This sample should be saved for analysis but should contain little Na+-K+-ATPase. Next a volume of elution buffer (Table 1) equal to 1.5 bed volumes was added to the top of the agarose, and the column was gently mixed end to end for 15 min at 37°C. The column was mounted vertically, and elution buffer was collected from the bottom of the column by gravity. Although not done in the experiments reported here, it is now recommended that an additional 0.5 bed volume of elution buffer be added to the top of the column and an equal volume collected at the bottom to elute protein that has been detached from the ouabain, but not washed off the column. Finally, 1.5 bed volumes of a solution containing 6% (wt/vol) SDS and 25 mM imidazole (pH 7.4) were added and collected. This last solution denatured and removed any bound protein. Again, it is recommended that an additional 0.5 bed volume of buffer be added to the top of the agarose and collected at the bottom to elute any remaining protein. In designing experiments with the column, one should appreciate that when the buffer is lowered to the top of the agarose, the volume of buffer on the column is equal to the bed volume. If protein is present under these conditions, the space occupied by the protein is approximately equal to one-half of the bed volume. The buffer space is larger, because the protein does not enter the agarose.

                              
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Table 1.   Solutions used for dog kidney Na+-K+-ATPase in Figs. 1 and 2

Purification Procedures

Na+-K+-ATPase can be purified from both microsomes and cells using either Na + ATP + Mg or high K to bind Na+-K+-ATPase to the column and either Na + ATP or Na+ ouabain to elute this enzyme. To show how the ouabain-affinity column may be used, this paper shows the complete purification of Na+-K+-ATPase from rat kidney microsomes using one set of conditions to bind (high K) and one set of conditions (Na + ATP) to elute (Fig. 3). The results in Fig. 4 show the amount of Na+-K+-ATPase eluted with Na + ATP or Na+ ouabain after Na+-K+-ATPase was purified, using either Na + ATP + Mg or K to bind Na+-K+-ATPase to the column. Purification of Na+-K+-ATPase from glomerulosa cells is illustrated using Na + ATP + Mg to bind Na+-K+-ATPase and Na + ouabain to elute (Fig. 5). In all of the purification experiments, the column itself was run as described above for previously purified dog kidney Na+-K+-ATPase, unless otherwise noted. The purification procedure takes ~2 h from the time loading buffer is added until purified Na+-K+-ATPase is eluted from the column. Note that the conditions and procedures described here may need to be modified when using the column to purify Na+-K+-ATPase from other kinds of preparations.

Rat kidney microsomes. The following is a general procedure for the purification of rat kidney Na+-K+-ATPase from microsomes with specific references to the experiments shown in Figs. 3 and 4. The column was run as described above for the dog kidney Na+-K+-ATPase, unless otherwise noted. A volume of 0.5 ml of rat kidney micorosomes containing ~3 mg protein suspended in homogenization buffer was added to an equal volume of double-strength loading buffer. For example, in the purification shown in Fig. 3, Na+-K+-ATPase was bound to the column with high K. Accordingly, the double-strength loading buffer contained 60 mM KCl, 2 mM EDTA, 50 mM imidazole, 4 µM leupeptin, and 1 mM PMSF, pH 7.4. SDS dissolved in loading buffer was slowly added to the microsome suspension to a final concentration of 0.4 mg SDS/mg protein in a volume of ~2 ml, containing ~1.4 mg protein/ml and ~0.58 mg SDS/ml. The final ratio of SDS to protein in this solution is important in terms of the purification (20), and the optimum ratio should be experimentally determined for each kind of preparation. After SDS was added, the solution was incubated with gentle shaking at room temperature for 30 min and then centrifuged at 4°C at 5,000 g for 3 min to remove debris that might subsequently impede flow through the column. Supernatant was removed and diluted 1:1 with loading buffer. This solution was then added to the ouabain-affinity matrix in the column. The column was capped and rotated end to end for 45 min at 4°C. The column was then mounted vertically, and gravity flow was initiated. The sample that immediately came off the column was collected as the pass-through fraction. With the use of gravity flow, the column was washed with loading buffer at 4°C and washed again at room temperature with the corresponding wash solution which contained CHAPS and phosphatidylcholine (Table 2 for Fig. 3). Note that the length of the wash and the concentration of CHAPS may need to be experimentally determined to define the ideal conditions for a particular preparation. Flow through the column was halted, elution buffer was added, and the column was rotated end to end at 37°C for either 15 min (Na + ATP elution) or 30 min (Na + ouabain elution). At the end of this period, the column was mounted vertically, and gravity flow was reinitiated and the sample collected. Subsequently, SDS elution buffer was added and collected.

                              
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Table 2.   Solutions used to purify rat kidney Na+-K+-ATPase in the procedure shown in Fig. 3

When Na+-K+-ATPase was purified using Na + ATP + Mg to bind the enzyme to the column (Fig. 4), the loading and washing buffers were similar to those shown in Table 3 for intact cells, except that the loading solution did not contain CHAPS or NaF. In the experiment shown in Fig. 4, the solution used to elute Na+-K+-ATPase with Na + ATP was the same as the eluting solution shown in Table 2. The ouabain eluting solution used in Fig. 4 was the same as that shown in Table 3. For purification with Na + ATP + Mg (Fig. 4), the column was first washed with loading buffer and then with a wash solution that was similar to the loading buffer, except that it contained CHAPS, phosphatidylcholine, and no SDS.

                              
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Table 3.   Solutions for the purification of the phosphorylated Na+-K+-ATPase from glomerulosa cells shown in Fig. 5

In attempting to purify Na+-K+-ATPase using K to bind the enzyme to the column, we initially used 150 mM K, but this concentration of K precipitated the SDS (29). Consequently, we reduced the K from 150 to 30 mM. Below 30 mM K, the amount of Na+-K+-ATPase that bound to the column was reduced. We chose a concentration of 30 mM K in which to run the column, because this concentration was high enough to get Na+-K+-ATPase to bind to the column without causing the SDS to precipitate.

Specific procedure for rat adrenal cells. Approximately 2 × 106 cells were washed in modified medium 199 (GIBCO) containing no phosphate, pH 7.4. The cells were suspended in 2 ml of the same medium containing 0.5 mCi [32P]orthophosphate and incubated at 37°C with gentle shaking. After 2 h, the cells were placed on ice, washed three times with the same medium 199 without 32P, and resuspended in 2 ml of the Na + ATP + Mg loading buffer (Table 3). The sample was then incubated at 37°C for 30 min and centrifuged at 5,000 g for 5 min, and the supernatant was loaded onto the column equilibrated with the same loading buffer. Unless otherwise noted, the column was run as described above for dog kidney enzyme. The column was incubated at 4°C for 45 min. Flow was reinitiated, and the pass-through sample was collected. The column was then rinsed with 10 bed volumes of the wash buffer at 4°C (Table 3) followed by 7 bed volumes at room temperature. Na+-K+-ATPase was eluted first with the ouabain eluting solution and then with the SDS eluting solution (Table 3).

Sample concentration. Eluted Na+-K+-ATPase was concentrated using a Centricon 30 (Amicon, Beverly, MA). Nonspecific protein loss was reduced by pretreating the upper chamber of the Centricon with 1% nonfat milk or adding 0.1% SDS to the sample before it was concentrated.

Procedure for Regenerating and Storing the Column

The column was washed with a solution containing 6% SDS, followed by 50 ml of a solution containing 50 mM imidazole, pH 7.4. The ouabain-affinity matrix was stored in a solution containing 50 mM imidazole, pH 7.4, and 0.05% sodium azide at 4°C.

Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis was carried out according to Laemmli (23). The stacking gel was 4.0%, and the running gel was 7.5% acrylamide. Samples were mixed with sample buffer, heated to 37°C for 15 min, and run overnight at a constant 55 V. Gels were stained with silver (9).

Protein Measurement

Total protein was measured by the method of Lowry et al. (25) with BSA as the standard. The amount of Na+-K+-ATPase eluted from the column was estimated from the amount of the alpha -subunit visualized on silver-stained gels prepared by means of SDS-PAGE. The amount of alpha -subunit was estimated using a Personal Densitometer and its ImageQuant software (Molecular Dynamics, Sunnyvale, CA) employing previously purified dog kidney Na+-K+-ATPase and/or phosphorylase B as a standard.

Na+-K+-ATPase Assay

Na+-K+-ATPase activity was measured at 37°C as ouabain-sensitive ATPase activity over a 30-min period in a volume of 0.3 ml as previously described (36). The assay buffer contained 130 mM NaCl, 20 mM KCl, 25 mM imidazole (pH 7.4), 3 mM ATP, 4 mM MgCl2, 0.5 mM EDTA, 0.3 mg/ml BSA, and 1 mM ouabain, where appropriate. Pi was quantified by the method of Forbush (13). Assay tubes contained 1-4 µg rat kidney membrane protein and <0.1 µg purified dog kidney Na+-K+-ATPase.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Ligand-Dependent Binding of Dog Kidney Na+-K+-ATPase

We first determined whether the ouabain-affinity column specifically bound Na+-K+-ATPase in the conformations one would predict from the known interactions of Na+-K+-ATPase with ouabain. Initial studies were performed with dog kidney Na+-K+-ATPase previously purified by the method of Jorgensen (20), which results in membrane sheets containing almost exclusively Na+-K+-ATPase. Na+-K+-ATPase prepared in this manner bound to the column in the presence of Na + ATP + Mg (Fig. 1). The combination of these three ligands put Na+-K+-ATPase into the E2-P conformation to which ouabain binds with high affinity (28). Na+-K+-ATPase did not bind in the presence of either Na or Na + ATP (Fig. 1), because they presumably fail to promote the formation of E2-P.


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Fig. 1.   Previously purified dog kidney Na+-K+-ATPase bound to ouabain affinity column as a function of Na, K, ATP, and Mg. Amount of Na+-K+-ATPase bound to column was calculated as difference between amount of alpha -subunit loaded on column and that which immediately passed through column. Amount of Na+-K+-ATPase was measured by densitometry of silver-stained gels containing aliquots of respective samples relative to known amounts of Na+-K+-ATPase. Because Na+-K+-ATPase used in this experiment had been previously purified, alpha -subunit was identified as single band that ran at 100 kDa. Composition of solutions is given in Table 1. At least 3 separate experiments were performed under each of 4 experimental conditions. Amounts bound are represented as means ± SE.

Na+-K+-ATPase also bound to the column in the presence of K (Table 1) alone (Fig. 1). Maximum binding to the column required at least 30 mM K and did not vary between 30 and 150 mM K (data not shown). These data are consistent with the observation that ouabain binds to Na+-K+-ATPase in the presence of high K (17). K may support ouabain binding, because it stabilizes E2 forms of the enzyme (16). Under other conditions, K competes with ouabain (14, 16) and lowers the rate of ouabain binding (18, 24). Thus our data suggest that dog kidney Na+-K+-ATPase in membrane sheets can bind to the ouabain-affinity column in both E2-P and in a complex with K, as predicted from the interaction of Na+-K+-ATPase with free ouabain. From these data we conclude that the ouabain-affinity column specifically binds Na+-K+-ATPase at the same site that it binds to untethered ouabain. The conformation-dependent binding of Na+-K+-ATPase to the ouabain-affinity column also suggests that the column may be useful for studying how Na+-K+-ATPase interacts with ouabain, although no one has yet demonstrated that the column is inherently superior to existing approaches for such studies.

In the experiments shown in Fig. 1, similar amounts of Na+-K+-ATPase (6-10 µg/ml bed vol) were added to columns with bed volumes that ranged in size from 1 to 2 ml. Most were 1.5 ml. In six similar experiments, 9.47 ± 0.38 µg Na+-K+-ATPase/ml bed volume were put on the column in the presence of Na + ATP + Mg. Of that amount, 72% bound to the column (Fig. 1). The mean amount of Na+-K+-ATPase added to the column in the presence of K was 8.42 ± 0.63 µg/ml bed volume of which 98% bound (Fig. 1). Thus these experiments suggest that the column can bind at least 8-10 µg Na+-K+-ATPase/ml bed volume.

Ligand-Dependent Elution of Dog Kidney Na+-K+-ATPase

The addition of excess free Na + ouabain eluted <= 5% of previously bound dog kidney Na+-K+-ATPase whether Na+-K+-ATPase was bound in the presence of Na + ATP + Mg or high K (Fig. 2A). The small amount eluted is consistent with the high affinity with which dog kidney Na+-K+-ATPase binds to ouabain (33). Na+-K+-ATPase not eluted with Na + ouabain was subsequently eluted with 6% SDS (Fig. 2A), which dissolves the membrane sheets and denatures Na+-K+-ATPase.


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Fig. 2.   Elution of dog kidney Na+-K+-ATPase. After Na+-K+-ATPase was bound to column using Na + ATP + Mg or K loading buffers, sample was eluted using 1 of 2 procedures. Either column was eluted with a solution containing 10 mM Na + ouabain (Ouab) followed by another solution containing 6% SDS and no Na + ouabain (A) or column was eluted with a solution containing Na + ATP, followed by a solution containing 6% SDS (B). Compositions of these solutions are given in Table 1. Amount of protein in each sample was measured as described in legend to Fig. 1. Amounts eluted are represented by means ± SE of at least 3 separate experiments.

On the other hand, the addition of Na + ATP, which reverses ouabain binding (19), eluted 22% of the dog kidney Na+-K+-ATPase bound in the presence of K and 8% of the enzyme bound in the presence of Na + ATP + Mg (Fig. 2B). Thus, for the dog kidney enzyme, which has a high affinity for ouabain, it is easier to elute Na+-K+-ATPase with Na + ATP than with Na + ouabain. All Na+-K+-ATPase not eluted with Na + ATP was recovered in the elution with SDS (Fig. 2B).

Purification of Rat Kidney Na+-K+-ATPase

The ouabain-affinity column was used to purify Na+-K+-ATPase from rat kidney microsomes with a concomitant increase in specific activity (Fig. 3). Although other kinds of ouabain-affinity columns have been developed (27, 30, 32, 35), this is the first successful purification of Na+-K+-ATPase achieved using a ouabain-affinity column.


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Fig. 3.   Purification of rat kidney Na+-K+-ATPase. A: silver-stained gel showing proteins present at different stages of purification. Enzyme was bound in presence of K (Table 2), and purification was carried out as described in MATERIALS AND METHODS starting with 3.34 mg rat kidney microsomes. An aliquot of sample applied to column is shown in lane 2. Lane 3 contains an aliquot of sample that immediately passed through column without binding. Aliquots of samples collected at end of wash at 4°C and room temperature are in lanes 4 and 5, respectively. Aliquots of samples eluted with Na + ATP and 6% SDS are shown in lanes 6 and 7, respectively. Large arrows in lanes 4 and 7 point to alpha -subunit clearly visible in lanes 2, 3, and 6. Small arrow in lane 7 points to beta -subunit faintly visible in lane 6. Lane 2 was loaded with 139 µg and lane 6 with 1.1 µg protein. Composition of solutions is given in Table 2. Lane 1 contains molecular mass markers as indicated. All samples off column were concentrated to same extent. B: 100-kDa region of proteins shown in A exposed to antisera to rat alpha 1-isoform of Na+-K+-ATPase. Lanes shown correspond to lanes 2-6 of A. C: Na+-K+-ATPase activity of eluted fractions corresponding to lanes 2-6 of A.

The purification procedure followed in the experiment shown in Fig. 3 is summarized here. Additional details are available in MATERIALS AND METHODS. The first step was to extract rat kidney microsomes in low concentrations of SDS at a specific ratio of SDS to protein for a specified period of time. This solution was then diluted to reduce the concentration of SDS and loaded onto the column. The composition of the loading buffer used in this experiment is given in Table 2. The column was washed with loading buffer and then with wash buffer containing 6 mM CHAPS and phosphatidylcholine (Table 2). Na+-K+-ATPase was eluted with the elution buffer (Table 2), which contained Na + ATP to reverse ouabain binding.

In the above procedure, Na+-K+-ATPase was purified by a combination of two detergent treatments. The SDS treatment was similar to the procedure developed by Jorgensen (20). In this case, low concentrations of SDS, which do not solubilize the membrane, are used to extract other proteins and leave Na+-K+-ATPase in its native phospholipid bilayer (20). In the Jorgensen procedure, Na+-K+-ATPase purified with SDS is collected on a sucrose gradient (20). We replaced the gradient with the ouabain-affinity column. In the Jorgensen method, the SDS treatment is sufficient to purify Na+-K+-ATPase (20). However, in our experiments, SDS only partially purified Na+-K+-ATPase, even though we varied the SDS-to-protein ratio over the same range (0.2-0.7) that others have successfully used to purify Na+-K+-ATPase to homogeneity. The higher ratios gave more pure enzyme but reduced the yields (data not shown). It is likely that the higher concentrations of SDS, which might have fully purified Na+-K+-ATPase, partially denatured Na+-K+-ATPase resulting in reduced binding to the column. A SDS-to-protein ratio of 0.4 was used in the experiment shown in Fig. 3. This ratio partially purified Na+-K+-ATPase and produced optimum yields from the column. The rest of the purification was achieved by the 6 mM CHAPS present in the wash solution (Table 2). This wash was so effective that it might be possible to purify Na+-K+-ATPase by omitting the SDS from the experiment entirely. We suggest that the wash with CHAPS dissolved the membranes and removed all proteins, except for Na+-K+-ATPase which remained specifically bound to the column. Phosphatidylcholine was included with the 6 mM CHAPS to prevent Na+-K+-ATPase from aggregating when the CHAPS concentration was reduced in the final elution (Table 2) to prevent this detergent from subsequently inhibiting Na+-K+-ATPase activity.

Purified Na+-K+-ATPase (Fig. 3A, lane 6) eluted from the column had a specific activity at least eightfold higher than the initial sample. The alpha -subunit (large arrows in Fig. 3A) was recognized by antisera to the alpha 1-isoform of the rat Na+-K+-ATPase (Fig. 3B). The eluted protein did not react with antisera raised to either the alpha 2- or alpha 3-isoforms (data not shown). In the experiment shown in Fig. 3, the amount of Na+-K+-ATPase added to the column (Fig. 3A, lane 2) exceeded the capacity of the column. Consequently, most of the protein immediately passed through without binding (Fig. 3A, lane 3) or changing in specific activity (Fig. 3C, sample corresponding to lane 3). After the column was washed at 4°C and then at room temperature, very little protein was eluted from the column (Fig. 3, lanes 4 and 5). The addition of Na + ATP eluted a total of 13.8 µg protein, of which an aliquot was applied to lane 6 (Fig. 3A). Because the bed volume in this experiment was 2 ml, there was ~7 µg of rat kidney Na+-K+-ATPase bound per milliliter bed volume, which was similar to the amount of dog kidney Na+-K+-ATPase that bound to the column. Little remaining protein was eluted with the subsequent addition of 6% SDS (Fig. 3, lane 7), indicating that the Na + ATP removed almost all of the previously bound Na+-K+-ATPase and that there were few other proteins still bound to the column after the addition of Na + ATP. In some other experiments, 6% SDS eluted a minor amount of Na+-K+-ATPase and significant amounts of other proteins (data not shown). These other proteins were present in the sample applied to the column and were probably bound to the column nonspecifically. The stability of the purified Na+-K+-ATPase is not yet known, because it was assayed on the day it was isolated.

The eightfold increase in specific activity that was achieved (Fig. 3C) gave a final value of 16 µmol · mg-1 · min-1, which is approximately one-half the value of the most active preparations of Na+-K+-ATPase purified by the method of Jorgensen (20), but well within the range of routine preparations obtained by this method. For instance, the dog kidney Na+-K+-ATPase used in the experiments shown in Figs. 1 and 2 that was purified by this method had a specific activity of 15 µmol · mg-1 · min-1.

According to the following analysis, Na+-K+-ATPase was probably purified more than 8-fold, perhaps close to 16-fold, depending in part on how one estimates the amount of Na+-K+-ATPase present in the original microsomes. On the basis of Na+-K+-ATPase activity, 6% of the total protein applied to the column was Na+-K+-ATPase. This calculation assumes all Na+-K+-ATPase in the sample was functioning at a maximum theoretical rate of 10,000 molecules ATP hydrolyzed/min (21) and that the molecular mass of Na+-K+-ATPase is 280 kDa (alpha beta alpha beta ) (31). If Na+-K+-ATPase was 6% of the initial protein, the alpha -subunit, which has a molecular mass of 100 kDa, would be 5/7 × 6% = 4.3% of the membrane protein. A scan of the protein in the sample loaded onto the column indicates that the protein at 100 kDa (band in lane 2 of Fig. 3A at level of large arrow) is 3% of the applied protein, in reasonable agreement with the calculated amount of the alpha -subunit. A scan of the silver-stained bands in the purified product (Fig. 3, lane 6) indicates that the alpha -subunit, which is the band of 100 kDa at the large arrow, represents 90% of the total protein visible in that lane. Even if Na+-K+-ATPase were 100% pure, the alpha -subunit should only be ~70% of the total protein, due to the presence of the beta -subunit which has a protein core of ~40 kDa but runs at ~60 kDa because of glycosylation. Although it is difficult to see in this silver-stained gel, a band corresponding to the beta -subunit is visible at the position of the small arrow in lane 6 of Fig. 3. A more prominent band is visible at this position when the purified product is displayed on gels stained with Coomassie blue (data not shown), suggesting that the beta -subunit stained better with Coomassie blue than with silver. Possible differences in the relative staining intensities of the alpha - and beta -subunits contribute to uncertainty of knowing exactly how much Na+-K+-ATPase was purified. Nevertheless, if Na+-K+-ATPase were 6% of the original membrane protein and the purification was eightfold, as suggested by the increase in specific activity, the Na+-K+-ATPase would be 48% of the membrane protein. In this case, the alpha -subunit should be 5/7 × 48% or 34% of the visible protein. Because the alpha -subunit is 90% of the visible protein, the purification was probably substantially greater than eightfold. If this is true, then some of the purified enzyme is probably not functioning at a maximum rate or is inactive.

Binding and Elution of Rat Kidney Na+-K+-ATPase

The total amount of rat kidney Na+-K+-ATPase recovered after purification was the same whether the enzyme was bound to the column in the presence of K or Na + ATP + Mg (Fig. 4). In these experiments, the enzyme was purified as described in the experiment shown in Fig. 3, except for differences in the specific ligands used to bind and elute Na+-K+-ATPase (see MATERIALS AND METHODS). We also tried to purify Na+-K+-ATPase by binding the enzyme to the column in the presence of Na or Na + ATP. However, no Na+-K+-ATPase was subsequently recovered when the column was eluted with either ouabain or Na + ATP (data not shown). The lack of recovered Na+-K+-ATPase is most likely because of the failure of Na+-K+-ATPase to bind to the column under these conditions (Fig. 1).


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Fig. 4.   Effect of Na + ouabain and Na + ATP on elution of rat kidney Na+-K+-ATPase from ouabain-affinity column. Na+-K+-ATPase was either bound in presence of Na + ATP + Mg or in presence of high K and purified as described in detail for experiment shown in Fig. 3 (see MATERIALS AND METHODS and corresponding tables for details on composition of solutions). A: column was eluted with a solution containing 10 mM Na + ouabain followed by another solution containing 6% SDS and no Na + ouabain. B: column was eluted with a solution containing Na + ATP followed by a solution containing 6% SDS. Total amount of Na+-K+-ATPase eluted in each group is sum of 2 treatments. Na+-K+-ATPase in microsomes was extracted with a low concentration of SDS, added to column (1.73-2.7 mg membrane protein/ml agarose), and further purified with CHAPS as explained in procedure outline for Fig. 3. Bed volumes of columns ranged from 1 to 2 ml; most were 1.5 ml. Amount of Na+-K+-ATPase eluted was measured as explained in legend to Fig. 1. In this case, alpha -subunit was assumed to be band that was eluted from column at same position as major band at arrow in Fig. 3A. Data presented are means ± SE of at least 3 separate experiments.

In contrast to dog kidney Na+-K+-ATPase, ouabain eluted significant amounts of rat kidney Na+-K+-ATPase from the column (Fig. 4A). The amount of rat kidney Na+-K+-ATPase eluted from the column with ouabain was the same whether Na+-K+-ATPase was applied to the column in the presence of Na + ATP + Mg or high K (Fig. 4A). The observation that Na + ouabain eluted significant amounts of rat kidney Na+-K+-ATPase and little dog kidney Na+-K+-ATPase is consistent with the reported lower affinity of the rat enzyme for ouabain. These data suggest that the column can be used to separate isoforms of Na+-K+-ATPase that vary in their affinity for ouabain.

In these experiments, Na + ATP eluted approximately one-half of the total Na+-K+-ATPase that bound to the column (Fig. 4B). The remainder of the bound Na+-K+-ATPase, along with some other proteins (data not shown), were eluted with 6% SDS (Fig. 4B). It is not known why Na + ATP did not elute all of the bound enzyme, as it did in other similar experiments (Fig. 3). One explanation is that original procedures used to elute Na+-K+-ATPase may have contributed to the variability. For instance, in the original procedure, 1.5 bed volumes of elution buffer were added to the agarose that was still wet with loading buffer. This is the equivalent of adding 1 bed volume of loading buffer to the 1.5 bed volumes of elution buffer. If the Na + ATP + Mg loading buffer contained 4 mM Mg and the elution buffer contained 4 mM EDTA, the final concentration would be 1.6 mM Mg and 2.4 mM EDTA. This ratio of EDTA to Mg should have been enough to chelate the Mg to the point that Na+-K+-ATPase could be eluted with Na + ATP, but not with a comfortable margin of safety to cover experimental variations. Columns loaded with K do not contain Mg, but in this case, the wash buffer left on the column would have contributed K to final mixture of buffers making the Na + ATP elution less effective. The second potential problem is that the original procedure used to collect the eluted protein may have left some of the protein on the column, making the Na + ATP and the Na + ouabain elutions appear less effective than they really were and increasing the amount of Na+-K+-ATPase that appeared to be eluted with SDS. We have corrected these potential problems in the new elution procedure given in the MATERIALS AND METHODS. Another potential explanation for the incomplete removal of Na+-K+-ATPase with either Na + ATP or Na + ouabain is that the enzyme in some preparations is heterogeneous. There is no direct evidence for heterogeneity, but it is possible that some Na+-K+-ATPase was denatured by the SDS.

The total amount of rat kidney Na+-K+-ATPase eluted (Fig. 4) was very similar to the amounts observed for dog kidney Na+-K+-ATPase (Fig. 2). This is significant, because the affinity of the dog kidney enzyme for ouabain at room temperature (33) is approximately two orders of magnitude higher than that of rat (1), although they both contain the alpha 1-isoform of Na+-K+-ATPase (7). With this difference in affinity, one might expect under some conditions that much more dog than rat Na+-K+-ATPase should bind to the column. However, in our experiments, we added much more rat than dog Na+-K+-ATPase to the columns, favoring the binding of more rat enzyme. Also, both enzymes were bound at 4°C and eluted at an elevated temperature. The lower temperature was chosen for binding and the higher temperature for elution, because the rate at which ouabain comes off Na+-K+-ATPase is known to be an exponential function of temperature (34). Thus we anticipated that Na+-K+-ATPase would bind more tightly in the cold and elute more easily at an elevated temperature. Lowering the binding temperature could also have reduced the difference in affinity between the rat and dog Na+-K+-ATPase, because the lower affinity of the rat enzyme for ouabain is primarily because of its higher off-rate for ouabain (1, 34). In designing experiments using the column, temperature is an important experimental variable.

Purification of Phosphorylated Na+-K+-ATPase From Rat Adrenal Glomerulosa Cells

To determine if the column could be used to purify phosphorylated Na+-K+-ATPase from cells with only modest amounts of Na+-K+-ATPase (15), we developed the appropriate procedures to isolate Na+-K+-ATPase from rat glomerulosa cells. The experiment shown here was done to purify phosphorylated Na+-K+-ATPase. Glomerulosa cells were loaded with [32P]orthophosphate, lysed in a solution containing both CHAPS and SDS (see MATERIALS AND METHODS), and purified on the ouabain-affinity column with CHAPS. An autoradiograph that shows the phosphoproteins loaded onto the column is shown in lane 1 of Fig. 5. The column was then washed and eluted first with Na + ouabain and then with SDS (Table 3). Na + ouabain eluted a phosphoprotein of 100 kDa (Fig. 5, lane 2). This protein is likely to be the alpha -subunit of Na+-K+-ATPase, because it is the expected size and it was specifically eluted with Na + ouabain. In addition, an eluted protein of this same molecular mass reacted with an antibody to the alpha -subunit of the rat alpha 1-isoform, but not those to the alpha 2- or alpha 3-isoforms (data not shown). The subsequent addition of 6% SDS (Fig. 5, lane 3) eluted a number of other phosphoproteins that were present in the sample that was applied to the column (Fig. 5, lane 1). In these experiments with intact cells, CHAPS was added to the lysing/loading buffer to immediately solubilize the cells and minimize any subsequent changes in phosphorylation. SDS may also have helped in solubilization, but its intended purpose was to help purify Na+-K+-ATPase as described for the kidney microsomes. The SDS-to-protein ratio was 0.2, one-half the value used for the rat kidney, because this ratio worked best in the presence of CHAPS. Na+-K+-ATPase purified from cells loaded with [32P]orthophosphate was not stained with either silver or Coomassie blue after SDS-PAGE due to the incorporated radioactivity.


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Fig. 5.   An autoradiograph showing recovery of phosphorylated Na+-K+-ATPase from rat adrenal glomerulosa cells. In this experiment, cells were loaded with [32P]orthophosphate, washed, extracted in CHAPS and SDS, and loaded onto column in presence of Na + ATP + Mg (lane 1). Column was then washed, and sample was eluted with a solution containing 10 mM Na + ouabain (lane 2) followed by 6% SDS (lane 3). Solutions used in these experiments are given in Table 3.

The level of phosphorylation of the alpha -subunit recovered after SDS-PAGE was not sensitive to 0.6 M hydroxylamine (4) (data not shown), indicating that the observed phosphorylation is distinct from the acyl phosphate formed during the enzymatic cycle of Na+-K+-ATPase equivalent to aspartic acid-369 (22). The recovery of phosphorylated alpha -subunit suggests that the Na+-K+-ATPase in adrenal glomerulosa cells is phosphorylated under basal conditions, as has been observed in other cells (5, 35). The site of this phosphorylation and its possible effect on the activity of Na+-K+-ATPase is not known.

Because Na+-K+-ATPase eluted with ouabain (Fig. 5) is inactive, we used Na + ATP in other experiments to elute active Na+-K+-ATPase. The amount of protein recovered was too small to calculate the resulting specific activity. Starting with 3 × 106 cells from eight rats, only ~0.5 µg Na+-K+-ATPase was obtained, which was enough to measure either activity or protein, but not both in the same experiment. When the samples were analyzed by SDS-PAGE, the resulting degree of purity was similar to the silver-stained gel shown in Fig. 3A for the rat kidney. Although we cannot be certain, our general impression was that the improvement in purity was not reflected in a corresponding increase in specific activity, suggesting that some of the enzyme may have been denatured.

Properties of the Ouabain-Affinity Matrix

The ouabain-affinity matrix described in this paper may function better than previously described forms for a number of reasons. First, the length of the spacer is 13 atoms, which evidently allows Na+-K+-ATPase to interact with tethered ouabain as it does with ouabain free in solution. Second, the covalent bond formed between the epoxy group and the ouabain may be unusually stable. We have used a single column 5-10 times without a decline in capacity. Also, we find that the amount of ouabain that leaches off the column to be very small, enabling one to measure the activity of the eluted Na+-K+-ATPase. According to literature available from the manufacturer, epoxide chemistry can immobilize ligands containing amino, thiol, and hydroxyl groups. Because amino and thiol groups are not present on ouabain, the epoxy group most likely couples to ouabain via its hydroxyl groups. Ouabain has eight hydroxyl groups of which three are on the sugar. It is likely that the epoxy group binds to all eight hydroxyl groups, but that only those ouabain molecules that are attached at the sugar are capable of binding Na+-K+-ATPase. This conclusion is suggested by the observation that strophanthidin, which is ouabain minus the sugar, binds well to Na+-K+-ATPase. Also, it is likely that the binding of the epoxy groups to any of the other OH groups would interfere with the ability of ouabain to bind to Na+-K+-ATPase. Given the number of epoxy groups (15-20 µmol/ml) available, the amount of Na+-K+-ATPase that binds to the ouabain affinity column appears low. For instance, if all epoxy groups bound a molecule of ouabain which in turn bound a molecule of Na+-K+-ATPase, one could theoretically have 20 µmol Na+-K+-ATPase bound/ml bed volume. Because Na+-K+-ATPase has a molecular mass of ~140 kDa (alpha beta alpha beta ), this would be equal to ~2 g alpha -subunit bound/ml bed volume, clearly far in excess of the ~10 µg/ml bed volume observed. According to the manufacturer, the coupling efficiency should be between 50 and 80% for most ligands, which in this case refers to the attachment of the epoxy groups to ouabain. In the experiments described in this paper, we measured the amount of Na+-K+-ATPase that bound to ouabain, not the amount of ouabain that was coupled to the column. The capacity of the column to bind Na+-K+-ATPase could be determined by the absolute number of ouabain molecules bound to agarose in a position to couple with Na+-K+-ATPase and/or to the number of ouabain molecules that have access to Na+-K+-ATPase embedded in membrane sheets. At this point, we have not distinguished between these two possibilities. Thus we do not know if the amount of enzyme recovered from the column is limited by the inherent capacity of the column or by how Na+-K+-ATPase is presented to the column.

In conclusion, a new ouabain-affinity column was developed that specifically binds and releases Na+-K+-ATPase as a function of the ligands (Na, K, ATP, and Mg) that control the binding of ouabain to Na+-K+-ATPase. The column is a tool that can be used to purify and study Na+-K+-ATPase. Using the column, we have developed procedures to purify active Na+-K+-ATPase from rat kidney microsomes and phosphorylated Na+-K+-ATPase from rat adrenal glomerulosa cells. This form of Na+-K+-ATPase from rat has a low affinity for ouabain, but the column also binds the high-affinity Na+-K+-ATPase from dog kidney. Because the amount of Na+-K+-ATPase that binds to the column appears low, the column appears best suited for purifying modest amounts (~10 µg/ml bed volume) of Na+-K+-ATPase from cells that contain average amounts of Na+-K+-ATPase. The advantage of using the column is that it can remove Na+-K+-ATPase from dilute suspensions and elute it in concentrated form. The column does not offer any obvious advantages over the Jorgensen method for purifying Na+-K+-ATPase from cells that contain large amounts of Na+-K+-ATPase. Thus use of the column compliments existing purification methods that are primarily useful for purifying Na+-K+-ATPase from cells that contain abundant amounts of Na+-K+-ATPase. Possible applications of the column include the purification of Na+-K+-ATPase from expression vectors and cells that contain average to small amounts of Na+-K+-ATPase. Purification of Na+-K+-ATPase from the latter cells will make it possible for the first time to examine the extent to which Na+-K+-ATPase is altered by posttranslational modifications in the majority of cells. The column can also be used to study how Na+-K+-ATPase interacts with ouabain, providing potential insights into the reaction mechanism of Na+-K+-ATPase. In this regard, we showed that Na+-K+-ATPase bound to the column in the presence of only K, confirming a previously reported, but generally unanticipated property of Na+-K+-ATPase. Finally, the same technology used here to couple ouabain to agarose could be modified to make a column that could be used to separate plasma membranes containing Na+-K+-ATPase (32) from intracellular membranes when both are released into the medium during cell fractionation.

    ACKNOWLEDGEMENTS

We thank Dr. Steve Cala for helpful discussions and Drs. Bliss Forbush, Joseph F. Hoffman, and Robert L. Post for reading and commenting on the manuscript.

    FOOTNOTES

This work was supported by grants from the American Heart Association of Michigan, by National Heart, Lung, and Blood Institute Grant HL-48885, and by the Vascular Biology Program of the Department of Internal Medicine at Wayne State University.

Address for reprint requests: D. R. Yingst, Dept. of Physiology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201.

Received 24 December 1997; accepted in final form 26 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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