Departments of 1 Physiology and 2 Internal Medicine, Division of Endocrinology, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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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 · mg1 · min
1.
Na+-K+-ATPase
purified from glomerulosa cells that were prelabeled with [32P]orthophosphate
was phosphorylated on the
-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
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INTRODUCTION |
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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
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).
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MATERIALS AND METHODS |
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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 · mg1 · 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
1-,
2-, and
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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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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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 theNa+-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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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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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
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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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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 -subunit (large
arrows in Fig. 3A) was recognized by
antisera to the
1-isoform of
the rat
Na+-K+-ATPase
(Fig. 3B). The eluted protein did
not react with antisera raised to either the
2- or
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 · mg1 · 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 () (31). If
Na+-K+-ATPase was 6% of the initial protein,
the
-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
-subunit. A scan of the silver-stained bands in the purified
product (Fig. 3, lane 6) indicates
that the
-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
-subunit should only be ~70% of the total
protein, due to the presence of the
-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
-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
-subunit stained better with Coomassie
blue than with silver. Possible differences in the relative staining
intensities of the
- and
-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
-subunit
should be 5/7 × 48% or 34% of the visible protein. Because the
-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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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 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
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The level of phosphorylation of the -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
-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 (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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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