The alpha -Subunit of the Colonic H+,K+-ATPase Assembles with beta 1-Na+,K+-ATPase in Kidney and Distal Colon*

Juan Codina, Juan T. Delmas-Mata, and Thomas D. DuBose Jr.Dagger

From the Division of Renal Diseases and Hypertension, Department of Internal Medicine, University of Texas, Houston Medical School, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Previous experiments from our laboratory (Codina, J., Kone, B. C., Delmas-Mata, J. T., and DuBose, T. D., Jr. (1996) J. Biol. Chem. 271, 29759-29763) demonstrated that the alpha -subunit of the colonic H+,K+-ATPase (HKalpha 2) requires coexpression with a beta -subunit to support H+/K+ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HKalpha 2 formed stable and functional alpha ·beta complexes when coexpressed with either the rat beta 1-subunit of the Na+,K+-ATPase or the beta -subunit of the gastric H+,K+-ATPase, suggesting that different beta -subunits may interact with HKalpha 2. The present studies tested this hypothesis by development and application of a specific antibody against HKalpha 2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HKalpha 2 co-precipitates with the same beta -subunit in organs known to express HKalpha 2 protein. The data demonstrate that HKalpha 2 assembles with beta 1-Na+,K+-ATPase in the renal medulla and in distal colon.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

It is assumed that all members of the X+,K+-ATPase family, including the gastric H+,K+-ATPase isoform, exist as dimers of alpha - and beta -subunits (1, 2). The alpha - or catalytic subunit of the H+,K+-ATPase has a molecular mass of approximately 100 kDa, contains the binding site for specific inhibitors such as Sch-28080 and omeprazole, and is responsible for internalizing K+ and secreting H+. The catalytic subunit for those X+,K+-ATPases characterized thus far requires, for biological activity, a glycosylated beta -subunit. Nevertheless, the absolute requirement of the colonic alpha  H+,K+-ATPase subunit (HKalpha 2)1 for a beta -subunit for functionality, has been called into question (3).

Lee and associates (3) reported that when the rat HKalpha 2 is expressed in a baculovirus expression system, in the absence of exogenous beta -subunit, the HKalpha 2 migrated to the plasma membrane and exhibited K+-ATPase activity that was partially sensitive to Sch-28080 and totally insensitive to ouabain. In contrast a different pharmacologic profile was observed when HKalpha 2 was expressed in oocytes from Xenopus laevis (4, 5). Cougnon and associates (4) employed the beta -subunit from toad bladder, for which there is no known mammalian counterpart. Our laboratory, on the other hand, used two different beta -subunits as follows: the beta -subunit of the rat gastric H+,K+-ATPase (beta HKalpha 1) and the beta 1-subunit of the rat Na+,K+-ATPase (beta 1) (5). Both studies reported an enzymatic activity, measured as 86Rb+ uptake, that was totally dependent on the presence of a beta -subunit. Also in contrast with the report of Lee and associates (3), this activity was insensitive to Sch-28080 and was partially sensitive to ouabain.

Although the findings of Lee and associates (3) suggested that under certain conditions HKalpha 2 may function in the absence of a beta -subunit, the study by Cougnon and associates (4) and that from our laboratory (5), cited above, demonstrated, nevertheless, that under certain physiological conditions different beta -subunits may interact with HKalpha 2. The interaction of HKalpha 2 with various beta -subunits observed in heterologous systems does not necessarily reveal what may pertain in intact tissues, however. Therefore, the purpose of this study was to analyze those interactions in the two tissues known to express HKalpha 2 protein (the rat distal colon and rat renal medulla) (6-8). Our results demonstrate that HKalpha 2 assembles with beta 1 in both organs.

    EXPERIMENTAL PROCEDURES
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Procedures
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Experimental Animals-- Chronic hypokalemia (LK) was accomplished in male Sprague-Dawley rats (135-175 g) by dietary K+ restriction (9). Rats in the LK group received tap water to drink and were maintained for 14 days on a customized vitamin-fortified nominally K+-free diet that contained 0.001 nmol/g potassium (ICN Biochemicals, Cleveland, OH, lot 960189). Control rats were pair fed each day with the same diet, to which KCl (0.03 mmol/g) was added. (ICN Biochemicals, P5411). Tap water was consumed ad libitum. The dietary modification was well tolerated and generated a stable and reproducible model of chronic K+ depletion. The rats were sacrificed in sets of two rats (one from each experimental group) to minimize bias during preparation.

Membrane Preparation-- To prepare membranes, the organs to be studied were homogenized with a Polytron (Brinkmann, model PT 10/35) in the presence of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM PMSF, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor (buffer A) containing 27% sucrose (w/v). Nuclei were removed by centrifugation at 2,000 × g for 4 min at 4 °C, and the supernatant was applied to the top of 45% (w/w) sucrose in buffer A and centrifuged at 200,000 × g for 45 min at 4 °C (10). The membranes in the interphase 27/45% sucrose were diluted in buffer A and collected by centrifugation at 25,000 × g. The final protein concentration was measured using the Lowry method (11).

Anti-HKalpha 2 Antibody-- A synthetic peptide (TPEQLDELLTNYQ) (12) that extends from amino acid 686 to 698 of the published sequence of HKalpha 2 (13) was synthesized (Genosys, The Woodlands, TX). The peptide was cross-linked to keyhole limpet hemocyanin. Two rabbits were injected and bled according to standard protocols. The peptide was tested for similarity to different X+,K+-ATPases, and the maximum identity (69%) of the peptide was with the human ATP1AL1 (or HKalpha 4) protein which is known to be expressed in brain, skin, and kidney (14), whereas the most divergence was with rat HKalpha 1 (23% identity) (15).

The sensitivity of the antibodies against HKalpha 2 were tested by Western blot analysis of HKalpha 2 synthesized "in vitro" using rabbit reticulocyte lysate as described previously by our laboratory (16). The specificity was tested using membranes prepared from rat stomach (enriched in HKalpha 1) (1), total rat kidney (enriched in alpha 1-Na+,K+-ATPase) (17), or rat distal colon (enriched in HKalpha 2) (6-8).

Immunoblots-- Membranes, prepared as described above, were separated on SDS-PAGE as described previously by our laboratory (18). The proteins were transferred overnight at 30 V to a nitrocellulose membrane (Schleicher and Schuell, BA85) in the presence of Tris/glycine buffer (25 mM Trizma (Tris base), 250 mM glycine, and 20% (v/v) methanol). The nonspecific binding sites of the nitrocellulose membrane were blocked in the presence of 5% nonfat dry milk in phosphate-buffered saline/Tween (10 mM sodium phosphate, pH 7.5, 150 mM NaCl, 0.05% Tween 20). This was followed by 2-h incubation with the primary antibody diluted 1:1000-10,000 in phosphate-buffered saline/Tween. After extensive washing, the membranes were incubated with a peroxidase-bound donkey anti-rabbit IgG, and the reacted antibody was detected using the ECL system (Amersham Pharmacia Biotech, RPN2108) following the manufacturer's instructions.

Synthesis of Recombinant Proteins-- The complete open reading frame of HKalpha 1 (15), HKalpha 2 (13), alpha 1-Na+,K+-ATPase (19), beta 1 (20), beta 2 (21), and beta HKalpha (22), all them from rat origin, were subcloned into the plasmid pAGA#2 as described previously by our laboratory (16). The in vitro synthesis of the recombinant proteins was accomplished using the TnT system (Promega, Madison, WI, catalog number L4610) in the presence of [35S]methionine following the instructions of the manufacturer. The quality of the synthesized proteins was verified by separating the synthesized proteins on SDS-PAGE, followed by autoradiography of the dried gel. The quantity of the synthesized protein was monitored by trichloroacetic acid precipitation of the 35S-synthesized recombinant protein (16).

Immunoprecipitation-- Subunit assembly was detected as coimmunoprecipitation of HKalpha 2 and a beta -subunit using an anti-HKalpha 2 antibody. Immune serum (150 µl) was incubated overnight at 4 °C with 1 mg (1 mg/ml) of membranes. The membranes were rinsed twice with 1 ml of buffer B (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM PMSF, 3 mM benzamidine, and 1 µg/µl soybean trypsin inhibitor). Membrane proteins were extracted with 500 µl of buffer B containing 1% CHAPS for 1 h at 4 °C. The insoluble material was removed by centrifugation, and the HKalpha 2/anti-HKalpha 2 antibody complex was precipitated by addition of 40 µl of protein A/G Plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number sc-2003) for 2 h at 4 °C (5). The resin was extensively washed, and the bound protein was extracted with Laemmli buffer (23) and separated in SDS-PAGE. The presence of a beta 1-subunit was detected with a monoclonal antibody (Upstate Biotechnology, Lake Placid, NY, catalog number 05-382).

Detection of Immunoprecipitated HKalpha 2 from Distal Colon Membranes-- After immunoprecipitation of HKalpha 2 from the distal colon with anti-HKalpha 2 antibody, the precipitated material was treated with Laemmli sample buffer containing 10% beta -mercaptoethanol and heated at 65 °C for 30 min. This treatment induces a total reduction of the dimer heavy/light chain of the IgGs that are similar in mobility to HKalpha 2. We found that about 60-70% of the HKalpha 2 is lost in this step at 65 °C.

Affinity of Anti-HKalpha 2 Antibody for Renal and Colonic HKalpha 2-- The interaction between anti-HKalpha 2 antibody and the native HKalpha 2 from different membranes was verified by incubating anti-HKalpha 2 antibody overnight at 4 °C in increasing concentrations of distal colon, renal medulla, and renal cortex membranes from both control and LK rats, in the presence of buffer B. The membranes were centrifuged, and the supernatant was used as a source of anti-HKalpha 2 antibody to detect HKalpha 2 in membranes prepared from distal colon (30 µg).

N-Deglycosylation of beta 1 with PNGase F-- To remove N-linked carbohydrates from the beta 1-subunit, membranes were incubated at 4 °C for 1 h in the presence of buffer B containing 1% CHAPS. The insoluble material was removed by centrifugation at 10,000 × g for 10 min at 4 °C, and deglycosylation was accomplished in the presence of 25,000 units/ml PNGase F (24) (New England Biolabs, Beverly, MA, catalog number 704L) for 1 h at 37 °C in the presence of 50 mM sodium phosphate, pH 8.0, 1 mM PMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and 1% CHAPS. The reaction was stopped by addition of Laemmli buffer (23); proteins were resolved on SDS-PAGE, and the immunoblots were performed as described above. When necessary, N-deglycosylation was performed using immunoprecipitated samples. In this case, deglycosylation was accomplished before the addition of Laemmli sample buffer to the Protein A/G Plus agarose, and the sample was incubated as described above for 60 min at 37 °C.

Deglycosylation of beta 1 Coimmunoprecipitated with HKalpha 2 from the Distal Colon-- The immunoprecipitated HKalpha 2/beta complex from the distal colon was incubated for 3 h at 37 °C in the presence of 20 mM sodium phosphate, pH 6.7, 1 mM PMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, 1% CHAPS, 0.03 units/ml endo-alpha -N-acetylgalactosaminidase (Sigma, catalog number E2391) (25), 0.07 units/ml neuraminidase (Sigma, catalog number N7885) (26), 0.05 units/ml alpha -L-fucosidase (Sigma, catalog number F7753) (27), and 25,000 units/ml PNGase F (New England Biolabs, Beverly, MA, catalog number 704L) (24). Incubation was stopped by addition of Laemmli sample buffer (23). The proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane, and beta 1 was detected by immunoblotting with the anti-beta 1 antibody exactly as described above. In some experiments the presence of either endo-alpha -N-acetylgalactosaminidase or PNGase F, or both were omitted during the deglycosylation (see figure legends).

Additional Reagents-- The monoclonal antibody against the beta 1-Na+,K+-ATPase was purchased from Upstate Biotechnology (Lake Placid, NY, catalog number 05-382). Anti-alpha 1-Na+,K+-ATPase (LEAVE) was a gift from Dr. T. Pressley (Texas Tech, Lubbock, TX) (28).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Antibody Specificity-- The specificity and sensitivity of the monoclonal beta 1-Na+,K+-ATPase antibody was verified using recombinant beta 1, beta 2, and beta HKalpha 1 synthesized in vitro (rabbit reticulocyte lysate) in the presence of [35S]methionine as described under "Experimental Procedures." The left panel of Fig. 1 demonstrates that anti-beta 1 antibody reacted with recombinant beta 1 (3.4 ng). Signal was not detected when either beta 2 (15 ng) or beta HKalpha 1 (16 ng) was applied to the SDS-PAGE. The ECL signal of the nitrocellulose membrane was allowed to decay for 24 h and exposed again overnight to detect [35S]methionine-labeled proteins. Fig. 1 (right panel) demonstrates that all three subunits ran as a single band of the expected mobility.


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Fig. 1.   Monoclonal anti-beta 1-Na+,K+-ATPase is specific for beta 1. Left panel, beta 1, beta 2, and beta HKalpha 1 were synthesized in rabbit reticulocyte in presence of [35S]methionine. The synthesized proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with the anti-beta 1-Na+,K+-ATPase antibody (1 µg/ml). Right panel, the ECL signal was allowed to decay for 24 h, and the nitrocellulose membrane was exposed overnight at room temperature. The symbols are as follows: beta 1, beta 1-subunit of the Na+,K+-ATPase; beta 2, beta 2-subunit of the Na+,K+-ATPase; beta HKalpha 1, beta -subunit of the gastric H+,K+-ATPase.

The specificity of anti-HKalpha 2 antibody was tested by immunoblot using rat plasma membranes from different organs as follows: distal colon membranes (10 µg) (enriched in HKalpha 2), whole kidney membranes (100 µg) (enriched in alpha 1-Na+,K+-ATPase), and stomach membranes (100 µg) (enriched in HKalpha 1). The results of one of these experiments are shown in Fig. 2, left panel. Specific antibody reactivity was detected in colon membranes (10 µg) but was not observed in kidney or stomach (up to 100 µg). This signal was blocked by preincubation (1 h at 4 °C) with the immunizing peptide (250 µM) (data not shown). By using recombinant protein as standard, the anti-HKalpha 2 antibody achieved a lower limit of detection equal to 2-5 ng. The specificity of the immunoprecipitation was established by immunoprecipitation of recombinant protein. The results of one experiment is shown in Fig. 2, right panel. Only HKalpha 2 was immunoprecipitated by the addition of anti-HKalpha 2 antibody, and the immunoprecipitation was blocked by incubating the antibody with immunizing peptide (data not shown).


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Fig. 2.   Anti-HKalpha 2 antibody is specific for HKalpha 2. Left panel, membranes from distal colon (5 µg), total kidney (100 µg), and stomach (100 µg) were resolved on SDS-PAGE, transferred to nitrocellulose membrane, and probed with the anti-HKalpha 2 antibody, diluted 1:1000. Right panel, alpha 1, HKalpha 1, and HKalpha 2 were synthesized in vitro in the presence of [35S]methionine using rabbit reticulocyte lysate. The synthesized protein was immunoprecipitated with 5 µl of immune serum. The immunoprecipitated samples were resolved on SDS-PAGE. The gel was dried and exposed overnight at room temperature. The symbols used are as follows: alpha 1, alpha 1-subunit of the Na+,K+-ATPase; HKalpha 1, alpha -subunit of the gastric H+,K+-ATPase; HKalpha 2, alpha -subunit of the colonic H+,K+-ATPase.

Heterogeneity of the beta 1-Subunit in the Renal Medulla and in the Distal Colon-- Our laboratory has reported previously that HKalpha 2 assembles stably and functionally in a heterologous expression system with two different rat beta -subunits (beta HKalpha 1 and beta 1) (5). To explore the relevance of this observation in vivo, membranes (1 mg) were prepared from the renal medulla of rats after chronic dietary K+ depletion (chronic hypokalemia), a condition known to maximize H+,K+-ATPase expression in kidney (6, 7). These membranes were incubated overnight with anti-HKalpha 2 antibody (150 µl) (see "Experimental Procedures"). The immunoprecipitated proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted against a monoclonal antibody to the beta 1-subunit of the rat Na+,K+-ATPase (Fig. 3, left panel). The anti-beta 1 monoclonal antibody detected a band at approximately 50-55 kDa (lane 1). This band was not present when the immunoprecipitation was performed with anti-HKalpha 2 antibody that was preincubated with the synthetic immunizing peptide (lane 2) or with preparations that did not contain membranes (lane 3). The central panel of Fig. 3 demonstrates that beta 1 was not detected from 1 mg of membranes prepared from the renal medulla of control rats. However, beta 1 was detected in the immunoprecipitated sample of renal medulla from control rats when as much as 35 mg of proteins were used in the immunoprecipitation experiment (data not shown). This observation agrees with data reported previously by our laboratory (6) and others (7, 8) that the abundance of HKalpha 2 is very low in renal medulla of control rats. Surprisingly, a beta 1-subunit was not detected, as well, when the immunoprecipitation experiments were performed using up to 1 mg of membranes from control distal colon or the distal colon harvested from rats with chronic hypokalemia (Fig. 3, right panel).


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Fig. 3.   Heterogeneity of the beta 1-subunit in the renal medulla and in the distal colon. HKalpha 2 was immunoprecipitated from 1 mg of membranes prepared as described under "Experimental Procedures." The presence of beta 1 in the immunoprecipitated sample was verified by immunoblot using a monoclonal antibody against the beta 1-subunit of the Na+,K+-ATPase. Left panel, membranes prepared from renal medulla of rats with chronic dietary K+ depletion (LK). Central panel, membranes prepared from renal cortex of control rats. Right panel, membranes prepared from distal colon of LK rats. Lane 1 of each panel: the immunoprecipitation was performed in the presence of the anti-HKalpha 2 antibody (150 µl). Lane 2 of each panel: the immunoprecipitation was performed with anti-HKalpha 2 antibody that had been preincubated in the presence of immunizing peptide (500 µM for 1 h at 4 °C). Lane 3 of each panel: the immunoprecipitation was performed in the absence of anti-HKalpha 2 antibody and in the absence of immune serum.

To verify that the distal colon contains a beta 1-subunit detectable by our monoclonal antibody, we performed the experiment displayed in Fig. 4. Distal colon membranes (50 µg) were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The beta 1-subunit was detected by immunoblot using the monoclonal beta 1 antibody. The antibody recognized a wide band in the region expanding from 40 to 80 kDa. This wide band "disappeared" upon deglycosylation with PNGase F (which remove the N-linked carbohydrates), generating a distinct band at 33 kDa (the expected mobility for beta 1). A band of the same mobility was generated upon deglycosylation of membranes from renal medulla (50 µg). The beta 1-subunit from both organs when deglycosylated displayed the same mobility as the beta 1-subunit synthesized in vitro using rabbit reticulocyte lysate (data not shown). Furthermore, when variable amounts of membranes were deglycosylated, it could be ascertained that there was approximately 2-fold more N-glycosylated beta 1 in the renal medulla as compared with the distal colon.


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Fig. 4.   beta 1 is present in distal colon membranes. Membranes from distal colon (50 µg) or from renal medulla (50 µg) were solubilized in the presence of CHAPS, and N-linked carbohydrates were removed with PNGase F as described under "Experimental Procedures." The presence of the beta 1-subunit was detected by immunoblot with the monoclonal antibody against the beta 1-Na+,K+-ATPase (1 µg/ml).

Anti-HKalpha 2 Antibody Recognizes Native HKalpha 2 in Distal Colon-- The observation that the beta 1-subunit was not immunoprecipitated from the distal colon using the anti-HKalpha 2 antibodies can be explained if HKalpha 2 were to undergo secondary modification in the distal colon which could render HKalpha 2 inaccessible to the antibody.

To test this possibility we performed two experiments. Increasing concentrations of distal colon membranes (from 0 to 2000 µg) were incubated overnight at 4 °C with anti-HKalpha 2 antibody. Membranes with bound antibody were then centrifuged, and the supernatant was used as a source of anti-HKalpha 2 antibody to detect HKalpha 2 by immunoblot in membranes prepared from distal colon. As expected, increasing concentrations of membranes decreased the intensity of the signal in the immunoblot, indicating that anti-HKalpha 2 antibody interacted with the native HKalpha 2 in the distal colon membranes (Fig. 5). In contrast, when 2000 µg of renal cortex from control rats were incubated with anti-HKalpha 2 antibody, the intensity of the band in the immunoblot did not decrease. This finding is in agreement with previous observations from our laboratory indicating that the levels of HKalpha 2 are either very low or absent from the renal cortex of normal rats (6). Finally, when the antibody was incubated overnight with 2000 µg of membranes prepared from renal medulla of chronically hypokalemic rats (condition shown to enhance the expression of HKalpha 2 in these membranes), there was a decrease in the intensity of the band on the immunoblot. This finding indicates that HKalpha 2 is present in membranes from the renal medulla during hypokalemia but, as expected, in less abundance than in the distal colon (6).


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Fig. 5.   Anti-HKalpha 2 antibody does not discriminate between the HKalpha 2 from distal colon and renal medulla. The indicated quantities of membranes were dissolved in 1 ml of buffer B (see "Experimental Procedures") and incubated overnight at 4 °C with immune serum (1 µl). The membranes were centrifuged, and the supernatant was used to detect HKalpha 2 in 30 µg of distal colon membranes. By using this assay the distal colon contains about 20-fold more HKalpha 2 than membranes prepared from renal medulla of rats with chronic dietary K+ depletion. These data are consistent with the ratio observed when levels of HKalpha 2 are quantified by direct immunoblotting in both types of membranes.

In the second experiment, distal colon plasma membranes (1 mg) were immunoprecipitated with anti-HKalpha 2 antibody. The pellet was dissolved in Laemmli sample buffer containing 10% beta -mercaptoethanol and incubated at 65 °C for 30 min. The sample was separated on SDS-PAGE, and the presence of HKalpha 2 was detected by immunoblot using the anti-HKalpha 2 antibody. Fig. 6 shows a band which comigrated with HKalpha 2 from distal colon membranes (5 µg). The mixture of anti-HKalpha 2 and anti-alpha 1 antibodies recognized a "doublet" in the membranes from distal colon, whereas a band corresponding to the mobility of HKalpha 2 was detected in the immunoprecipitated sample (data not shown). These findings verify that the immunoprecipitated alpha -subunit is not the alpha 1-Na+,K+-ATPase.


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Fig. 6.   Anti-HKalpha 2 antibody immunoprecipitates HKalpha 2 from the distal colon. HKalpha 2 from the distal colon was immunoprecipitated as described under "Experimental Procedures." The pellet was resuspended in Laemmli sample buffer containing 10% beta -mercaptoethanol. The sample was heated for 30 min at 65 °C and run on a SDS-PAGE, transferred to a nitrocellulose membrane, and HK2 detected by immunoblot.

Taken together, the two experiments described above indicate that anti-HKalpha 2 antibody does not discriminate between HKalpha 2 from renal medulla and distal colon, and the differences in the beta 1-subunit detected in the immunoprecipitation shown in Fig. 3 reside in the beta -subunit per se.

The beta 1 Associated with HKalpha 2 Is an N-Linked Carbohydrate in the Renal Medulla-- Fig. 4 reveals that it was more difficult for the beta 1 monoclonal antibody to detect the glycosylated as opposed to the deglycosylated form of beta 1 in distal colon. To investigate the possibility that beta 1 might be associated with HKalpha 2 in the distal colon, but could not be detected by Western analysis, we performed the following experiment. The anti-HKalpha 2 antibody was used to immunoprecipitate the HKalpha 2/beta complex from distal colon and renal medulla from rats after chronic dietary K+ depletion. The immunoprecipitated protein was N-deglycosylated in the presence of PNGase F and applied to SDS-PAGE and transferred to a nitrocellulose membrane. The presence of beta 1-subunit was tested with the monoclonal antibody against beta 1. The results shown in Fig. 7 demonstrate that the antibody detected beta 1 in the immunoprecipitate from renal medulla but not in the immunoprecipitate prepared from distal colon membranes.


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Fig. 7.   The beta 1 associated with HKalpha 2 is an N-linked carbohydrate in the renal medulla. Immunoprecipitation of HKalpha 2 from membranes (1 mg) prepared from renal medulla of LK rats and deglycosylation with PNGase F generated a sharp beta 1 band, which was not present when the same immunoprecipitation was performed using distal colon membranes (1 mg).

We have estimated, using recombinant HKalpha 2 as a standard (16), that each milligram of membranes from renal medulla contains approximately 30 ng of native HKalpha 2 (which would be predicted to associate with 10-12 ng of beta 1). By using recombinant beta 1 as standard, we detected that the total beta 1 immunoprecipitated from 1 mg of membranes from the renal medulla was approximately 10 ng. This finding suggests that in the renal medulla of the chronically hypokalemic rat, beta 1 may represent the only beta -subunit that associates with HKalpha 2. Comparison of the quantities of HKalpha 2 present in membranes from both renal medulla and distal colon indicates that the distal colon contains 20-fold more HKalpha 2 than the renal medulla. Nevertheless, beta 1 could not be detected in distal colon if deglycosylation was confined to conventional N-deglycosylation.

The previous observation that beta 1 did not immunoprecipitate with HKalpha 2 in the distal colon could be interpreted as follows: (a) in the distal colon, the beta 1 associated with HKalpha 2 is an O-linked carbohydrate; (b) in the distal colon, the beta 1 associated with the HKalpha 2 is an N-linked carbohydrate, but the glycosylated protein is not accessible to PNGase F; or (c) the beta 1-subunit does not associate with HKalpha 2 in the distal colon. As displayed in Fig. 8, the immunoprecipitated sample from the distal colon was deglycosylated with a mixture of enzymes to remove sialic acid, fucose, and O- and N-linked carbohydrates as described under "Experimental Procedures." The sample was separated on SDS-PAGE and transferred to a nitrocellulose membrane, and the presence of beta 1 was detected with anti-beta 1 monoclonal antibody. Fig. 8 shows that beta 1 was detected in the immunoprecipitated sample and, as in the renal medulla, displayed a mobility which was identical to the mobility of beta 1-Na+,K+-ATPase synthesized in vitro using rabbit reticulocyte lysate (data not shown).


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Fig. 8.   beta 1 associates with HKalpha 2 in the distal colon. HKalpha 2 from 1 mg of distal colon membranes was immunoprecipitated with anti-HKalpha 2 antibody. The immunoprecipitated HKalpha 2·beta complex was deglycosylated with a mixture of neuraminidase, alpha -L-fucosidase, endo-alpha -N-acetylgalactosaminidase, and PNGase F (see "Experimental Procedures"), and beta 1 was detected with the anti-beta 1 antibody. Left lane, no immunoprecipitated sample (enzymes alone); central lane, the immunoprecipitated sample that was not deglycosylated; right lane, the sample was deglycosylated with the enzyme mixture.

To determine if the beta 1-Na+,K+-ATPase associated with HKalpha 2 in the distal colon contained N- or O-linked carbohydrate, we performed the following experiment. The immunoprecipitated HKalpha 2/beta 1 complex was deglycosylated with PNGase F or endo-alpha -N-acetylgalactosaminidase to remove N- or O-linked carbohydrates, respectively. Proteins were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with the monoclonal antibody against the beta 1-subunit. The results demonstrated that beta 1 was not detected (Fig. 9, lanes 1 and 2). However, removal of the sialic acid and fucose with a mixture of neuraminidase and alpha -L-fucosidase generated a band that was recognized by the anti-beta 1-monoclonal antibody. This band displayed a mobility of 32 kDa (lane 3). Addition of endo-alpha -N-acetylgalactosaminidase to the mixture of neuraminidase and alpha -L-fucosidase, to remove the O-linked carbohydrates (lane 4), did not alter the mobility of the beta 1-subunit recognized by the monoclonal antibody. However, addition of PNGase F to the mixture of neuraminidase and alpha -L-fucosidase (lane 5) rendered a beta 1-subunit with the same mobility of the beta 1-subunit synthesized in vitro using rabbit reticulocyte lysate (data not shown), and this band also displayed a mobility that was identical to the band generated from the renal medulla after PNGase F treatment.


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Fig. 9.   The beta 1 associated with HKalpha 2 is an N-linked carbohydrate in the distal colon. HKalpha 2 from 1 mg of distal colon membranes was immunoprecipitated with anti-HKalpha 2 antibody. The immunoprecipitated sample was deglycosylated with endo-alpha -N-acetylgalactosaminidase (lane 1), PNGase F (lane 2), neuraminidase and alpha -L-fucosidase (lane 3), neuraminidase, alpha -L-fucosidase, and endo-alpha -N-acetylgalactosaminidase (lane 4), and neuraminidase, alpha -L-fucosidase, endo-alpha -N-acetylgalactosaminidase, and PNGase F (lane 5). The arrow labeled "beta 1" indicates the predicted mobility of beta 1-subunit synthesized in rabbit reticulocyte lysate. (Numbers to the right indicate the molecular mass in kilodaltons.)

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Our results clearly demonstrate that under physiological conditions HKalpha 2 associates with beta 1-Na+,K+-ATPase in both organs, the renal medulla and the distal colon. The present study employed immunoprecipitation techniques to investigate the specificity of association of HKalpha 2 with beta -subunits in the kidney and in the distal colon. Specifically, two questions were explored as follows: does HKalpha 2 associate with a beta -subunit in tissues in which HKalpha 2 protein is known to be expressed, and does HKalpha 2 associate with the same beta -subunit at these locations? Our data demonstrate that HKalpha 2 consistently co-precipitated with the beta 1-Na+,K+-ATPase subunit in the renal medulla and in the distal colon. The difference between both organs was in the complexity of the glycosylation, particularly in the distal colon, which necessitated complete deglycosylation removing sialic acid and/or fucose bound to the beta 1 to achieve deglycosylation by PNGase F.

The highly complex pattern of glycosylation of the beta 1-subunit coimmunoprecipitated with HKalpha 2 in the distal colon was the most likely explanation for our inability to detect the beta 1-subunit from immunoprecipitated samples from distal colon prior to more complete deglycosylation (Fig. 3, right panel, lane 1).

Lee and associates (3) have expressed HKalpha 2 in a baculovirus expression system in the absence of an exogenous beta -subunit. These investigators reported a K+-ATPase that was resistant to ouabain and partially sensitive to Sch-28080. These data raised the possibility that, under certain conditions, HKalpha 2 may be biologically active in the absence of a beta -subunit. Our data from distal colon and renal medulla do not support this hypothesis, since beta 1 was consistently coimmunoprecipitated with HKalpha 2 from both tissues.

Both, beta 1-Na+,K+-ATPase (29) and HKalpha 2 (3) have been localized to the apical membrane of distal colonocytes. Furthermore, our laboratory has demonstrated, using oocytes from X. laevis, that beta 1 assembles stably with HKalpha 2. The functionality of HKalpha 2/beta 1 was substantiated by 86Rb+(K+) uptake (5). These data suggested that in the distal colon beta 1 could represent the beta -subunit that associated with HKalpha 2. The findings of the present study establish that beta 1 and HKalpha 2 assemble stably and functionally in a plasma vesicle fraction from distal colon. More importantly, by establishing that the Na+,K+-ATPase interacts with HKalpha 2 in the renal medulla and in the distal colon, our data suggest additionally that a unique "colonic" beta -subunit may not exist. Factors that regulate alpha 1-Na+,K+-ATPase/beta 1 and HKalpha 2/beta 1 association in cells containing both HKalpha 2 and alpha 1-Na+,K+-ATPase remain to be defined.

    ACKNOWLEDGEMENTS

We thank Thomas A. Pressley (Texas Tech, Lubbock, TX) and Carlos H. Pedemonte (University of Houston, Houston) for carefully reading the manuscript and for their suggestions during the course of this study.

    FOOTNOTES

* This work was supported in part by NIDDKD Grant DO-30603-16 from the National Institutes of Health (to T. D. D., Jr.).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.

Dagger To whom correspondence should be addressed: Division of Renal Diseases and Hypertension, University of Texas-Houston Medical School, 6431 Fannin St., Room 4.148, Houston, TX 77030. Tel.: 713-500-6868; Fax: 713-500-6882; E-mail: tdubose{at}heart.med.uth.tmc.edu.

1 The abbreviations used are: HKalpha 2, alpha -subunit of the colonic H+,K+-ATPase; HKalpha 1, alpha -subunit of the gastric H+,K+-ATPase; beta HKalpha 1, beta -subunit of the gastric H+,K+-ATPase; beta 1, beta -subunit of the Na+,K+-ATPase; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PMSF, phenylmethylsulfonyl fluoride.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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