From the Division of Renal Diseases and Hypertension, Department of
Internal Medicine, University of Texas, Houston Medical School,
Houston, Texas 77030
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
-subunit of the colonic
H+,K+-ATPase (HK
2) requires
coexpression with a
-subunit to support H+/K+ transport in a heterologous expression
system (Xenopus laevis oocytes). In these studies,
HK
2 formed stable and functional
·
complexes
when coexpressed with either the rat
1-subunit of the
Na+,K+-ATPase or the
-subunit of the gastric
H+,K+-ATPase, suggesting that different
-subunits may interact with HK
2. The present studies
tested this hypothesis by development and application of a specific
antibody against HK
2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HK
2 co-precipitates with the same
-subunit in organs
known to express HK
2 protein. The data demonstrate that
HK
2 assembles with
1-Na+,K+-ATPase in the renal
medulla and in distal colon.
 |
INTRODUCTION |
It is assumed that all members of the
X+,K+-ATPase family, including the
gastric H+,K+-ATPase isoform, exist as dimers
of
- and
-subunits (1, 2). The
- 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
-subunit.
Nevertheless, the absolute requirement of the colonic
H+,K+-ATPase subunit
(HK
2)1 for a
-subunit for functionality, has been called into question (3).
Lee and associates (3) reported that when the rat HK
2 is
expressed in a baculovirus expression system, in the absence of
exogenous
-subunit, the HK
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
HK
2 was expressed in oocytes from Xenopus laevis (4, 5). Cougnon and associates (4) employed the
-subunit
from toad bladder, for which there is no known mammalian counterpart.
Our laboratory, on the other hand, used two different
-subunits as
follows: the
-subunit of the rat gastric
H+,K+-ATPase (
HK
1) and the
1-subunit of the rat
Na+,K+-ATPase (
1) (5). Both
studies reported an enzymatic activity, measured as
86Rb+ uptake, that was totally dependent on the
presence of a
-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 HK
2 may function in the absence of a
-subunit, the study by Cougnon and associates (4) and that from our
laboratory (5), cited above, demonstrated, nevertheless, that under
certain physiological conditions different
-subunits may interact
with HK
2. The interaction of HK
2 with
various
-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 HK
2 protein (the rat
distal colon and rat renal medulla) (6-8). Our results demonstrate
that HK
2 assembles with
1 in both
organs.
 |
EXPERIMENTAL PROCEDURES |
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-HK
2 Antibody--
A synthetic peptide
(TPEQLDELLTNYQ) (12) that extends from amino acid 686 to 698 of the
published sequence of HK
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
HK
4) protein which is known to be expressed in brain,
skin, and kidney (14), whereas the most divergence was with rat
HK
1 (23% identity) (15).
The sensitivity of the antibodies against HK
2 were
tested by Western blot analysis of HK
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 HK
1)
(1), total rat kidney (enriched in
1-Na+,K+-ATPase) (17), or rat
distal colon (enriched in HK
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 HK
1 (15), HK
2 (13),
1-Na+,K+-ATPase (19),
1 (20),
2 (21), and
HK
(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 HK
2 and a
-subunit using an
anti-HK
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 HK
2/anti-HK
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
1-subunit was
detected with a monoclonal antibody (Upstate Biotechnology, Lake
Placid, NY, catalog number 05-382).
Detection of Immunoprecipitated HK
2 from Distal
Colon Membranes--
After immunoprecipitation of HK
2
from the distal colon with anti-HK
2 antibody, the
precipitated material was treated with Laemmli sample buffer containing
10%
-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 HK
2. We found
that about 60-70% of the HK
2 is lost in this step at
65 °C.
Affinity of Anti-HK
2 Antibody for Renal and
Colonic HK
2--
The interaction between
anti-HK
2 antibody and the native HK
2 from
different membranes was verified by incubating anti-HK
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-HK
2
antibody to detect HK
2 in membranes prepared from distal
colon (30 µg).
N-Deglycosylation of
1 with PNGase F--
To
remove N-linked carbohydrates from the
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
1 Coimmunoprecipitated with
HK
2 from the Distal Colon--
The immunoprecipitated
HK
2/
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-
-N-acetylgalactosaminidase (Sigma, catalog number
E2391) (25), 0.07 units/ml neuraminidase (Sigma, catalog number N7885)
(26), 0.05 units/ml
-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
1 was detected by immunoblotting with the
anti-
1 antibody exactly as described above. In some
experiments the presence of either
endo-
-N-acetylgalactosaminidase or PNGase F, or both were
omitted during the deglycosylation (see figure legends).
Additional Reagents--
The monoclonal antibody against the
1-Na+,K+-ATPase was purchased
from Upstate Biotechnology (Lake Placid, NY, catalog number 05-382).
Anti-
1-Na+,K+-ATPase (LEAVE) was
a gift from Dr. T. Pressley (Texas Tech, Lubbock, TX) (28).
 |
RESULTS |
Antibody Specificity--
The specificity and sensitivity of the
monoclonal
1-Na+,K+-ATPase
antibody was verified using recombinant
1,
2, and
HK
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-
1
antibody reacted with recombinant
1 (3.4 ng). Signal was
not detected when either
2 (15 ng) or
HK
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- 1-Na+,K+-ATPase is specific
for 1. Left panel, 1,
2, and HK 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- 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: 1,
1-subunit of the Na+,K+-ATPase;
2, 2-subunit of the
Na+,K+-ATPase; HK 1,
-subunit of the gastric H+,K+-ATPase.
|
|
The specificity of anti-HK
2 antibody was tested by
immunoblot using rat plasma membranes from different organs as follows: distal colon membranes (10 µg) (enriched in HK
2),
whole kidney membranes (100 µg) (enriched in
1-Na+,K+-ATPase), and stomach
membranes (100 µg) (enriched in HK
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-HK
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 HK
2 was immunoprecipitated
by the addition of anti-HK
2 antibody, and the
immunoprecipitation was blocked by incubating the antibody with
immunizing peptide (data not shown).

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Fig. 2.
Anti-HK 2 antibody is specific
for HK 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-HK 2 antibody, diluted 1:1000.
Right panel, 1, HK 1, and
HK 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: 1, 1-subunit of the
Na+,K+-ATPase; HK 1, -subunit
of the gastric H+,K+-ATPase;
HK 2, -subunit of the colonic
H+,K+-ATPase.
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|
Heterogeneity of the
1-Subunit in the Renal Medulla
and in the Distal Colon--
Our laboratory has reported previously
that HK
2 assembles stably and functionally in a
heterologous expression system with two different rat
-subunits
(
HK
1 and
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-HK
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
1-subunit of the rat
Na+,K+-ATPase (Fig.
3, left panel). The
anti-
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-HK
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
1 was not detected from 1 mg of
membranes prepared from the renal medulla of control rats. However,
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 HK
2 is very low
in renal medulla of control rats. Surprisingly, a
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 1-subunit
in the renal medulla and in the distal colon. HK 2
was immunoprecipitated from 1 mg of membranes prepared as described
under "Experimental Procedures." The presence of 1
in the immunoprecipitated sample was verified by immunoblot using a
monoclonal antibody against the 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-HK 2 antibody (150 µl). Lane 2 of each
panel: the immunoprecipitation was performed with
anti-HK 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-HK 2 antibody and in the
absence of immune serum.
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To verify that the distal colon contains a
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
1-subunit was detected by
immunoblot using the monoclonal
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
1). A band of
the same mobility was generated upon deglycosylation of membranes from renal medulla (50 µg). The
1-subunit from both organs
when deglycosylated displayed the same mobility as the
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
1 in the renal medulla as compared with the distal
colon.

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Fig. 4.
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
1-subunit was detected by immunoblot with the monoclonal
antibody against the
1-Na+,K+-ATPase (1 µg/ml).
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Anti-HK
2 Antibody Recognizes Native
HK
2 in Distal Colon--
The observation that the
1-subunit was not immunoprecipitated from the distal
colon using the anti-HK
2 antibodies can be explained if
HK
2 were to undergo secondary modification in the distal
colon which could render HK
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-HK
2 antibody. Membranes with bound antibody were then centrifuged, and the
supernatant was used as a source of anti-HK
2 antibody to
detect HK
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-HK
2 antibody interacted with the native
HK
2 in the distal colon membranes (Fig.
5). In contrast, when 2000 µg of renal
cortex from control rats were incubated with anti-HK
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 HK
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 HK
2 in these
membranes), there was a decrease in the intensity of the band on the
immunoblot. This finding indicates that HK
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-HK 2 antibody does not
discriminate between the HK 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 HK 2
in 30 µg of distal colon membranes. By using this assay the distal
colon contains about 20-fold more HK 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 HK 2 are quantified by direct immunoblotting in
both types of membranes.
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In the second experiment, distal colon plasma membranes (1 mg) were
immunoprecipitated with anti-HK
2 antibody. The pellet was dissolved in Laemmli sample buffer containing 10%
-mercaptoethanol and incubated at 65 °C for 30 min. The sample
was separated on SDS-PAGE, and the presence of HK
2 was
detected by immunoblot using the anti-HK
2 antibody. Fig.
6 shows a band which comigrated with
HK
2 from distal colon membranes (5 µg). The mixture of
anti-HK
2 and anti-
1 antibodies recognized
a "doublet" in the membranes from distal colon, whereas a band
corresponding to the mobility of HK
2 was detected in the
immunoprecipitated sample (data not shown). These findings verify that
the immunoprecipitated
-subunit is not the
1-Na+,K+-ATPase.

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Fig. 6.
Anti-HK 2 antibody
immunoprecipitates HK 2 from the distal colon.
HK 2 from the distal colon was immunoprecipitated as
described under "Experimental Procedures." The pellet was
resuspended in Laemmli sample buffer containing 10%
-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.
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Taken together, the two experiments described above indicate that
anti-HK
2 antibody does not discriminate between
HK
2 from renal medulla and distal colon, and the
differences in the
1-subunit detected in the
immunoprecipitation shown in Fig. 3 reside in the
-subunit per
se.
The
1 Associated with HK
2 Is an
N-Linked Carbohydrate in the Renal Medulla--
Fig. 4 reveals that it
was more difficult for the
1 monoclonal antibody to
detect the glycosylated as opposed to the deglycosylated form of
1 in distal colon. To investigate the possibility that
1 might be associated with HK
2 in the
distal colon, but could not be detected by Western analysis, we
performed the following experiment. The anti-HK
2
antibody was used to immunoprecipitate the HK
2/
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
1-subunit was tested with the monoclonal antibody
against
1. The results shown in Fig.
7 demonstrate that the antibody detected
1 in the immunoprecipitate from renal medulla but not in
the immunoprecipitate prepared from distal colon membranes.

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Fig. 7.
The 1 associated with
HK 2 is an N-linked carbohydrate in the renal
medulla. Immunoprecipitation of HK 2 from membranes
(1 mg) prepared from renal medulla of LK rats and deglycosylation with
PNGase F generated a sharp 1 band, which was not present
when the same immunoprecipitation was performed using distal colon
membranes (1 mg).
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We have estimated, using recombinant HK
2 as a standard
(16), that each milligram of membranes from renal medulla contains approximately 30 ng of native HK
2 (which would be
predicted to associate with 10-12 ng of
1). By using
recombinant
1 as standard, we detected that the total
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,
1
may represent the only
-subunit that associates with
HK
2. Comparison of the quantities of HK
2
present in membranes from both renal medulla and distal colon indicates
that the distal colon contains 20-fold more HK
2 than the
renal medulla. Nevertheless,
1 could not be detected in
distal colon if deglycosylation was confined to conventional
N-deglycosylation.
The previous observation that
1 did not
immunoprecipitate with HK
2 in the distal colon could be
interpreted as follows: (a) in the distal colon, the
1 associated with HK
2 is an
O-linked carbohydrate; (b) in the distal colon,
the
1 associated with the HK
2 is an
N-linked carbohydrate, but the glycosylated protein is not
accessible to PNGase F; or (c) the
1-subunit
does not associate with HK
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
1 was detected with anti-
1 monoclonal antibody. Fig. 8 shows that
1 was detected in the
immunoprecipitated sample and, as in the renal medulla, displayed a
mobility which was identical to the mobility of
1-Na+,K+-ATPase synthesized
in vitro using rabbit reticulocyte lysate (data not
shown).

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Fig. 8.
1 associates with
HK 2 in the distal colon. HK 2 from 1 mg of distal colon membranes was immunoprecipitated with
anti-HK 2 antibody. The immunoprecipitated
HK 2· complex was deglycosylated with a mixture of
neuraminidase, -L-fucosidase,
endo- -N-acetylgalactosaminidase, and PNGase F (see
"Experimental Procedures"), and 1 was detected with
the anti- 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.
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To determine if the
1-Na+,K+-ATPase associated with
HK
2 in the distal colon contained N- or
O-linked carbohydrate, we performed the following
experiment. The immunoprecipitated
HK
2/
1 complex was deglycosylated with
PNGase F or endo-
-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
1-subunit. The results demonstrated that
1 was not detected (Fig.
9, lanes 1 and 2).
However, removal of the sialic acid and fucose with a mixture of
neuraminidase and
-L-fucosidase generated a band that
was recognized by the anti-
1-monoclonal antibody. This
band displayed a mobility of 32 kDa (lane 3). Addition of
endo-
-N-acetylgalactosaminidase to the mixture of
neuraminidase and
-L-fucosidase, to remove the
O-linked carbohydrates (lane 4), did not alter
the mobility of the
1-subunit recognized by the
monoclonal antibody. However, addition of PNGase F to the mixture of
neuraminidase and
-L-fucosidase (lane 5)
rendered a
1-subunit with the same mobility of the
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 1 associated with
HK 2 is an N-linked carbohydrate in the
distal colon. HK 2 from 1 mg of distal colon
membranes was immunoprecipitated with anti-HK 2 antibody.
The immunoprecipitated sample was deglycosylated with
endo- -N-acetylgalactosaminidase (lane 1),
PNGase F (lane 2), neuraminidase and
-L-fucosidase (lane 3), neuraminidase,
-L-fucosidase, and
endo- -N-acetylgalactosaminidase (lane 4), and
neuraminidase, -L-fucosidase,
endo- -N-acetylgalactosaminidase, and PNGase F (lane
5). The arrow labeled " 1" indicates
the predicted mobility of 1-subunit synthesized in
rabbit reticulocyte lysate. (Numbers to the right
indicate the molecular mass in kilodaltons.)
|
|
 |
DISCUSSION |
Our results clearly demonstrate that under physiological
conditions HK
2 associates with
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 HK
2 with
-subunits in the kidney and
in the distal colon. Specifically, two questions were explored as
follows: does HK
2 associate with a
-subunit in
tissues in which HK
2 protein is known to be expressed,
and does HK
2 associate with the same
-subunit at
these locations? Our data demonstrate that HK
2
consistently co-precipitated with the
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
1 to achieve
deglycosylation by PNGase F.
The highly complex pattern of glycosylation of the
1-subunit coimmunoprecipitated with HK
2
in the distal colon was the most likely explanation for our inability
to detect the
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 HK
2 in a
baculovirus expression system in the absence of an exogenous
-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,
HK
2 may be biologically active in the absence of a
-subunit. Our data from distal colon and renal medulla do not support this hypothesis, since
1 was consistently
coimmunoprecipitated with HK
2 from both tissues.
Both,
1-Na+,K+-ATPase (29) and
HK
2 (3) have been localized to the apical membrane of
distal colonocytes. Furthermore, our laboratory has demonstrated, using
oocytes from X. laevis, that
1 assembles
stably with HK
2. The functionality of
HK
2/
1 was substantiated by
86Rb+(K+) uptake (5). These data
suggested that in the distal colon
1 could represent the
-subunit that associated with HK
2. The findings of
the present study establish that
1 and
HK
2 assemble stably and functionally in a plasma vesicle
fraction from distal colon. More importantly, by establishing that the
Na+,K+-ATPase interacts with HK
2
in the renal medulla and in the distal colon, our data suggest
additionally that a unique "colonic"
-subunit may not exist.
Factors that regulate
1-Na+,K+-ATPase/
1
and HK
2/
1 association in cells containing
both HK
2 and
1-Na+,K+-ATPase remain to be
defined.
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.