From the Department of Biochemistry, McGill
University, Montreal, Quebec H3G 1A4, Canada and the
¶ Department of Biochemistry, Weizmann Institute of Science,
Rehovot 76100, Israel
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ABSTRACT |
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The functional role of the The Na,K-ATPase is the sodium pump protein responsible for
maintaining the electrochemical gradient present across the membranes of most animal cells (1). It consists of at least two subunits, A small single-transmembrane protein called the In this report we show that expression of Antibodies--
5'-RACE and pREP4- Transfections, Tissue Culture, and Membrane
Preparations--
HEK-293 cells at 50% confluency in a 14-cm culture
plate were transfected with pREP4 or pREP4- Western Blots--
Western blot analysis and densitometry
measurements were carried out as described previously (14) with the
following modifications. 10% polyacrylamide gels were run on a Protean
II gel electrophoresis apparatus (Bio-Rad), transferred to
polyvinylidene difluoride membranes (Millipore), and blotted with 6H
antibodies and Enzyme Assays--
Na,K-ATPase and Na-ATPase assays were carried
out at 37 °C in a final volume of 100 µl as described previously
(12). For Na,K-ATPase assays, final concentrations of reactants were:
100 mM NaCl, 10 mM KCl, 40 mM
choline chloride, 4 mM MgSO4, 1 mM
EDTA, 30 mM Tris-HCl (pH 7.4), and varying concentrations
of ATP as indicated. Na,K-ATPase activities shown represent the ATPase
activities inhibited by 5 mM ouabain and ranged from 1500 to 4500, 130 to 180, and 110 to 130 nmol Pi/mg/min for
kidney, HEK-pREP4, and HEK-pREP4- We showed previously that the Expression of the
Efforts to express
The blots shown in Fig. 2 indicate that
the Functional Effects of
To maximize the inhibitory effect of the antiserum, particularly for
tests of the effect of
We first compared the effect of The successful transfection of the Recently, Béguin et al. (7) have shown that the rat
The N-terminal sequence of the rat The presence of two distinct bands of Overall, our results suggest an interaction between the Na,K-ATPase and
the C-terminal tail of the The increase in apparent affinity for ATP effected by subunit of the
Na,K-ATPase was studied using rat
cDNA-transfected HEK-293
cells and an antiserum (
C33) specific for
. Although the sequence
for
was verified and shown to be larger (7237 Da) than first
reported, it still comprises a single initiator methionine despite the
expression of a
C33-reactive doublet on immunoblots. Kinetic
analysis of the enzyme of transfected compared with control cells and
of
C33-treated kidney pumps shows that
regulates the apparent
affinity for ATP. Thus,
-transfected cells have a decreased
K'ATP as shown in measurements of (i)
K'ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 µM ATP.
Consistent with the behavior of
-transfected cells,
C33
pretreatment increases K'ATP of the kidney
enzyme and K+ inhibition (1 µM ATP) of both
kidney and
-transfected cells. These results are consistent with
previous findings that an antiserum raised against the pig
subunit
stabilizes the E2(K) form of the enzyme
(Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634).
Overall, our data demonstrate that
is a tissue (kidney)-specific
regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-
antiserum.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
, each of which exists as one of several isoforms (
1,
2,
3, and
4 and
1,
2, and
3; for review,
see Ref. 2). The
subunit, also known as the catalytic subunit,
contains the binding sites for the enzyme's nucleotide and cation
substrates, as well as the catalytic and regulatory
(calcium-dependent and cAMP-dependent protein
kinase C and A, respectively) phosphorylation sites. The role of the
subunit is less clear, but it is required for normal processing and
expression of the enzyme and may have a role in regulating the
interaction of cations with the
subunit (3). The different isoforms
of the pump are expressed in a tissue- and development-specific fashion
and are believed to be distinct in both function and modes of
regulation (2).
subunit was
originally believed to be a third subunit of the pump. It was discovered by Forbush et al. (4) in 1978 and later cloned in rat, mouse, cow, sheep (5), human (6), and Xenopus
laevis (7); it has sequence homology to a family of
channel-inducing peptides (8-10). Although its function has remained
elusive, experiments in Xenopus oocytes have shown that the
subunit alters the K+ affinity of the pump in a
voltage- and Na+-dependent fashion (7) and may
induce cation channel activity (11). Our recent Western blot analysis
using an anti-
antiserum indicated that
protein is detected only
in the kidney medulla but not in other tissues tested (red blood cells,
heart, brain, and kidney glomerulus) including cultured cell lines
derived from cells of the kidney tubule. We showed that the antibodies
bound to the cytoplasmic tail of
and stabilized the
E2 form of the enzyme, presumably by disrupting
-
interactions (12).
in cells devoid of this
protein results in a significant increase in apparent affinity for ATP
and that the
-transfected cells resemble the
1
1
kidney enzyme in that this effect
is abrogated by antiserum raised against a 10-residue peptide of the C
terminus of the
subunit.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C33 is a rabbit polyclonal antiserum raised
against a peptide representing the C-terminal 10 amino acids of the
subunit. In the experiments reported herein,
C33 was used, and a
control nonimmune serum was obtained from the same rabbit prior to
immunization. The peptide, KHRQVNEDEL, was synthesized at the Alberta
Peptide Institute, University of Alberta, and used either as the free peptide for competition studies or linked to keyhole limpet hemocyanin and emulsified with Freund's adjuvant before injection into rabbits. Antibody 6H is a mouse monoclonal antibody specific for the
1 isoform of the Na,K-ATPase, and was a generous gift
from Dr. Michael Caplan, Yale University. Horseradish
peroxidase-labeled secondary antibodies (donkey anti-rabbit) were
purchased from BIO/CAN Scientific.
Synthesis--
5'-Rapid amplification
of cDNA ends (5'-RACE)1
was carried out using CLONTECH Marathon-ready
cDNA from rat kidney following the manufacturer's instructions.
Appropriate primers (see below) were synthesized, and the
subunit
gene sequence was amplified by polymerase chain reaction. The 5'-end
primer contained a site for HindIII endonuclease (boldface),
a Kozak sequence (underlined; see Ref. 13), and the first 24 bases of
the
subunit gene as determined by 5'-RACE
(GGGGGGGAAGCTTGCCGCCACCATGACAGAGCTGTCAGCTAACCAT). The 3'-end primer contained a BamHI endonuclease site
(boldface) and bases complementary to the last 24 bases of the
subunit gene as determined by Mercer et al. (5)
(GGGGGGGATCCGTCACAGCTCATCTTCATTGACCT). The resulting DNA was
then cleaved with these endonucleases and ligated into the
corresponding sites of pREP4 vector (Invitrogen) to make pREP4-
.
Sequencing of the recombinant plasmid was carried out using a Pharmacia
T7 sequencing kit. pREP4 and pREP4-
DNA used for transfections were
purified using Qiagen affinity columns according to the manufacturer's instructions.
using FuGENE 6 reagent
(Roche Molecular Biochemicals) and following the manufacturer's
instructions. Cells were selected for 10 days, divided among 5 × 14 cm plates, and allowed to grow to confluency (about 3 weeks) in
Dulbecco's modified Eagle's medium containing 10% newborn calf serum
and 400 µg/ml hygromycin B. Cellular membranes from transfected cells and from rat kidney medulla were prepared by the procedure described elsewhere (12).
C33 antiserum, both at dilutions of 1:10,000.
membranes, respectively. For
Na-ATPase assays, final concentrations of reactants were: 20 mM NaCl, 20 mM choline chloride, 2 mM MgSO4, 1 mM EDTA, 5 mM EGTA, 20 mM histidine-Tris (pH 7.4), and 1 µM ATP. To determine effects of K+ on
Na-ATPase, choline chloride was replaced by the indicated concentrations of KCl. For assays of effects of anti-
, membranes were preincubated for 1 h at 4 °C in the presence of immune
(
C33) or nonimmune (preimmune) sera at a ratio of 1:100. For
experiments of K inhibition of Na-ATPase, the sera were dialyzed for
48 h at 4 °C against three changes of 1000 volumes of 5 mM imidazole (pH 7.4). When present, the 10-mer peptide was
used in the antiserum preincubation at a concentration of 20 µM. K'ATP values were calculated by analyzing ATP activation curves using the Michaelis-Menten formulation. All experiments shown are representative of at least three
separate experiments, and each data point shown is the mean ± S.E. of the difference between triplicate determinations carried out in
the absence and presence of ouabain.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit protein is expressed in
a tissue-specific manner. Of the various rat tissues analyzed by
Western blotting (kidney medulla, kidney glomerulus, red blood cells,
heart, and axolemma),
was detected only in the kidney medulla (12).
In more recent experiments (not shown) this analysis has been extended
to additional tissues of the rat, namely the lung, small intestine,
stomach, and spleen. The
protein could not be detected in these
tissues except for a trace amount in the spleen (relative to
,
amounting to
2% of that present in the kidney medulla). The
kidney-specific presence of
also holds true with mouse tissues
(kidney, axolemma, and heart) analyzed similarly.2
Subunit in Mammalian Cells--
Our earlier
evidence of a modulatory role for the
subunit on the conformational
equilibrium of the Na,K-ATPase reaction was inferred from studies of
the effects of an anti-
antiserum on enzymatic activity. To evaluate
directly the functional role of
, it was essential to transfect
cDNA encoding
into mammalian cells devoid of
. An additional
goal of such experiments was to establish the basis for the existence
of
as a doublet in the rat (5) as it is in the Xenopus
kidney (7). Accordingly, we first used 5'-RACE to ascertain that the
previously reported cDNA of the rat
subunit comprised the
full-length sequence and, if not, whether the doublet in Western blots
is secondary to the presence of an additional start codon in the
mRNA for the
subunit as is the case for Xenopus
kidney (7). The resulting sequence, shown in Fig.
1, confirmed the presence of a single
initiator methionine. However, the
cDNA thus obtained encodes a
protein of 66 rather than 58 residues, as originally reported (5), and
corresponds to the sequence subsequently revised by Minor et
al. (11). The calculated molecular mass is 7237 Da. The dichotomy may be the result of either a cloning artifact or, possibly, an isoform
variant.3
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Fig. 1.
5'-Untranslated and coding regions of
rat subunit cDNA and deduced amino acid
sequence. The nucleotide sequence was determined by 5'-RACE
analysis as described under "Experimental Procedures."
in HeLa and HEK cells using a standard stable
transfection system resulted in levels of expression that, compared
with the kidney, were considered too low (
:
0.1) given the
relatively modest effects of anti-
on the kidney enzyme. In an
effort to increase the level of expression, we used the plasmid pREP4
that combines the advantages of "classical" transient and stable
expression systems. In addition to a hygromycin resistance gene, this
plasmid contains an origin of replication that allows it to remain
expressed episomally for several weeks in the nuclei of primate and
canine cells. Thus, hygromycin can be used to select for cells that
contain multiple copies of the gene (rather than just one).
Accordingly, we subcloned the gene for
(revised sequence shown in
Fig. 1) in pREP4 and transfected HEK-293 cells with both recombinant
and wild type plasmids. Membranes were made from the transfected
HEK-pREP4-
and control HEK-pREP4 cells, and the amount of
subunit protein relative to
subunit protein was estimated by a
comparison with kidney membranes using Western blot analyses of both
the
and
subunits.
doublet is present in both kidney and HEK-pREP4-
membranes
but not in control HEK-pREP4 membranes. The densities of the
subunit doublet and
subunit band of HEK-pREP4-
were compared
with those of the kidney using several dilutions and varying times of
exposure to film. We determined that pREP4-
membranes contain
34 ± 12% (S.E.) of the amount of
present in the kidney after
normalizing for
1 densities. Assuming that the
:
ratio of kidney is 1:1 (7, 15, 16), this indicates that the
stoichiometry of the
:
proteins in HEK-pREP4-
1:3. That
this ratio reflects
associated with
was confirmed in Western
blots of immunoprecipitates using the antibody 6H (not shown).
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Fig. 2.
Western blot analysis of rat kidney,
HEK-pREP4- , and HEK-pREP4 membranes.
Immunoblotting was carried out as described under "Experimental
Procedures." Lane 1, 2.0 µg of rat kidney membranes;
lane 2, 30 µg of HEK-pREP4-
membranes; lane
3, 30 µg of HEK-pREP4 membranes.
--
We showed earlier that binding of
antibodies raised to the
polypeptide doublet associated with the
pig kidney Na,K-ATPase binds to the cytoplasmic tail of the
subunit
(12). This binding was associated with partial inhibition of the
Na,K-ATPase activity. Moreover, inhibition varied as a function of
conditions that affect the rate-limiting step(s) during steady-state
hydrolysis, for example varying pH. Thus, the inhibition (
30%)
observed under conditions of optimal concentrations of substrates and
at pH 7.4 decreased as the pH level increased and increased as pH
decreased. We concluded that the antiserum caused a shift in the
E1
E2(K) equilibrium
toward E2(K).
in the transfected cells in which the
:
ratio is lower than in the kidney medulla, we tested the
prediction that inhibition would be greater at suboptimal ATP
concentrations, under which conditions the
E2(K)
E1 sequence becomes even more rate-limiting (17). For these experiments, a
10-residue peptide representing the C terminus of the
subunit was
synthesized and used for the production of
C33 antisera, allowing
confirmation of the specificity of the anti-
effects and providing
free 10-mer peptide for competition studies. Fig. 3A shows a representative
experiment on the effects of anti-
(serum
C33) on Na,K-ATPase
activity of renal enzyme at near saturating (1 mM) and
subsaturating (10 µM) concentrations of ATP. As
predicted, inhibition increases as the ATP concentration is lowered,
from 36 ± 4% inhibition at 1 mM ATP to 70 ± 11% at 10 µM ATP (averages of several experiments). In
addition, the presence of excess amounts of free peptide corresponding
to the C terminus of
during the preincubation reversed completely
the inhibition observed at both ATP concentrations; no effect on the
activity of nonimmune serum-treated enzyme was observed. Fig.
3B is a Lineweaver-Burk plot of a representative experiment
showing the effect of ATP concentration on activity. It shows that
pretreatment of the enzyme with antiserum
C33 caused a 1.8-fold
increase in K'ATP (for values, see
inset in Fig. 4). Vmax for
C33-treated enzyme was 78 ± 7% that for nonimmune serum-treated enzyme. The critical implication
of this result is that anti-
reverses an increase in affinity
effected by the
subunit. This hypothesis was tested in
HEK-pREP4-
cells and HEK-pREP4 cells.
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Fig. 3.
Effect of C33
antiserum and
C33-reactive peptide on ATP
affinity of renal pumps. A, rat renal membranes were
assayed for Na,K-ATPase activity at 100 µM or 1 mM ATP after preincubation in the presence of
C33
antiserum or nonimmune rabbit serum and in the absence or presence of
peptide representing the C-terminal 10 amino acids of the
subunit
(used to generate
C33). Differences between nonimmune and immune
serum-treated enzyme are significant (p < 0.01 using
Student's t test).
, nonimmune serum;
, nonimmune
serum + peptide;
,
C33;
,
C33 + peptide. B, rat renal membranes were assayed for Na,K-ATPase
activity (v) at different ATP concentrations after
preincubation in the presence of
C33 (open circles) or
nonimmune serum (filled circles). Lineweaver-Burk plots of a
representative experiment are shown.
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Fig. 4.
Effect of C33 on ATP
affinity of HEK-pREP4 and HEK-pREP4-
pumps. Membranes isolated from HEK-pREP4-
(filled
circles) and HEK-pREP4 (open circles) were assayed for
Na,K-ATPase activity (v) at varying ATP concentrations.
Lineweaver-Burk plots of a representative experiment are shown, with
Vmax values of the two membrane preparations
normalized to 1.0. Inset, a table summarizing
K'ATP values for
C33-treated and nonimmune
serum-treated kidney enzymes and HEK-pREP4-
and HEK-pREP4 enzymes.
IS,
C33; NIS, nonimmune serum. Differences
between nonimmune and immune serum-treated renal enzyme
(p < 0.01), as well as between pREP4- and
pREP4-
-transfected cells (p < 0.02), are
significant using Student's t test.
C33 on HEK-pREP4-
, HEK pREP4
cells, and kidney enzymes, all assayed at 10 µM ATP. The
experiment (not shown) indicated that
C33 caused 33 ± 2 and
82 ± 15% inhibition of HEK-pREP4-
and kidney enzymes,
respectively, and had no effect on the activity of HEK-pREP4 cells.
This inhibition is consistent with the aforementioned relative amounts
of
in kidney versus HEK-pREP4-
cells. Experiments
were then carried out to determine whether the
subunit has any
effect on K'ATP. The plots shown in Fig. 4
indicate that the HEK-pREP4-
enzyme has a significantly higher
affinity for ATP compared with control HEK-pREP4 enzyme (for
K'ATP values, see inset). The
-mediated-1.3-fold decrease in K'ATP in these
cells, although modest, is in fact similar to the effect of
in the
kidney membranes, taking into account the lower
:
ratio in the
transfected cells (approximately one-third that of kidney membranes).
This being the case, we used a more sensitive assay of ATP affinity to
magnify the effect of
and to determine whether anti-
antiserum
can reverse its effects. This assay takes advantage of the fact that
K+ inhibits Na-ATPase activity at a very low (1 µM) ATP concentration under which condition the (low
affinity) ATP-activated K+ deocclusion reaction becomes
rate-limiting. Accordingly, this inhibition decreases as the affinity
for ATP at its low affinity binding site increases (18). As shown in
Fig. 5A, K+ is
less effective at inhibiting the Na-ATPase activity of pumps of
-transfected membranes than of control membranes. Experiments were
then carried out to test and compare K+ inhibition, and the
effect of anti-
thereupon, of the enzyme of the kidney medulla,
HEK-pREP4-
, and HEK-pREP4. Fig. 5B shows the percentage
inhibition at 0.2 mM KCl of these pumps in the presence of
nonimmune versus immune serum. Whereas preincubation of
kidney and pREP4-
pumps with
C33 effected 2.1- and 1.5-fold increases in K+ inhibition, respectively, no
C33-mediated change was detected for HEK-pREP4 pumps.
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Fig. 5.
K+ inhibition of Na-ATPase
activity of rat kidney, HEK-pREP4, and HEK-pREP4-
pumps and the effect of
C33.
Membranes were assayed for Na-ATPase activity in varying concentrations
of KCl as described under "Experimental Procedures." A,
K+ inhibition profile of pREP4 (open circles)-
and pREP4-
(filled circles)-transfected cells.
B, inhibition of Na-ATPase activity in the presence of 0.2 mM KCl after preincubation with
C33 or nonimmune serum.
Differences between nonimmune and immune serum-treated kidney
(p < 0.01) and HEK-pREP4-
enzymes
(p < 0.02) are significant using Student's
t test.
, nonimmune serum;
,
C33.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit into mammalian
cells with sodium pumps devoid of this subunit has enabled the direct
analysis of the functional role of this Na,K-ATPase-associated protein.
Although the
subunit does not appear to be necessary for normal
Na,K-ATPase activity (7, 15, 19), its role as a modulator of function
is consistent with its appearance in a tissue (kidney)-specific manner.
subunit lowers the affinity of the pump for K+ in
cRNA-injected Xenopus oocytes, at least in the absence of Na+. A
-mediated decrease in
K'ATP could explain this increase in K'0.5 for K+ because, as a first
approximation, ATP and K+ affinities are inversely related
(20). However, that result may be confounded by the use of cRNA
synthesized using the original sequence for rat
(5). In a recent
report, the human
subunit was shown to induce cation channel
activity in Xenopus oocytes (11), consistent with several
reports of other channel-inducing membrane peptides (8, 9, 21). These
proteins have homology with the
subunit but are generally larger,
and some contain possible protein kinases C and A phosphorylation sites
at their C-terminal ends that are not present in the
subunit (5,
8-10). Although we have no information regarding such a role in our
transfected cells, it should be pointed out that the sequence of the
putative human
subunit reported in the aforementioned study
contains 30 extra amino acids at its N terminus (11) that are absent in
rat
(cf. Fig. 1). Whether the rat
subunit also has a
channel function and/or this extended N terminus confers a particular functional role in forming channels in Xenopus oocytes
remains to be determined.
subunit reported here and by
Minor et al. (11) is different from the one originally reported (5). That it is the correct sequence is substantiated by the
following observations. First, the
subunit doublet present in
membranes of transfected cells corresponds in size to that of kidney
membranes (Fig. 2). Second, the presence of a lysine residue at
position 13 (Fig. 1) where a glutamine was originally reported (5) is
in accordance with the finding that the upper band of the rat
subunit is cleaved by trypsin (treatment of intact right-side-out
microsomes (12)). Third, preliminary results using matrix-assisted
laser desorption ionization time-of-flight (MALDI-TOF) mass
spectroscopy indicate that the pig kidney
subunit has a length of
between 64 and 67 residues,4
consistent with a length of 66 residues reported here and in Ref.
11.
has been the subject of some
controversy. Whereas Mercer et al. first showed that a
single RNA species could yield two protein products evidenced on
Western blots using an artificial translation system (5), Béguin
et al. (7) showed that in X. laevis the two bands
were secondary to the presence of two distinct start codons (7). Our
results with 5'-RACE analysis preclude the presence of distinct ATG
codons for the rat protein, indicating instead that post-translational modifications are involved, because transfection of HEK-293 cells with
a gene containing single start and stop codons yielded two bands of
similar mobilities to those of the kidney
subunit. In addition,
preliminary mass spectroscopy results are consistent with the notion
that the difference between the two bands is the result of
post-translational modifications.4 The differences in the
ratio of the densities of the upper to the lower band between
subunits of kidney and transfected HEK membranes (see Fig. 2) suggest
tissue-specific variations in post-translational modifications. Whether
each band has some distinct role remains to be determined.
subunit that regulates ATP affinity and
that is reversible upon binding of antibodies to
. The finding that
increases the apparent affinity for ATP in
-transfected cells is
completely concurrent with the effect of anti-
on the
pump
of the kidney tubule. Moreover, under conditions in which
K+ sensitivity of Na-ATPase at low ATP concentration is
used as a sensitive marker of differences in ATP affinity, the reversal of the
effect by anti-
is similar with the enzyme of
-transfected cells and the kidney medulla. These similarities
underscore our earlier interpretation of the effect of
from
analysis of the effects of the anti-
antiserum. Whether the
increased apparent affinity for ATP is, in fact, a true increase in
affinity or a reflection of an alteration in conformational equilibrium
toward E1 form(s) is unclear and requires
further analysis, as does the question of whether the difference in ATP
affinity can be evidenced in a change in apparent affinity for
extracellular K+. Whatever the case, it is the change in
ATP affinity that is likely to be of major physiological relevance.
is
approximately 2-fold, as evidenced in either the effect of anti-
on
the kidney enzyme or of
transfected into HEK cells, extrapolating the ratio of
:
in HEK-pREP-
to that of the kidney. Such a
change in apparent affinity may be of critical physiological
importance. Although other physiological functions may be served by the
subunit (as suggested recently by Jones et al. (22)), an
almost 2-fold shift in ATP affinity is a potentially important
regulatory mechanism. The
subunit may serve to preserve the pumping
activity in cells or conditions in which the ATP level falls suddenly. Relevant to this notion is the observation that the renal outer medulla
is highly prone to anoxia because it works on the brink of anoxia even
in normal circumstances (23, 24). That the
subunit effect is
reversible upon addition of anti-
antibodies further underscores its
physiological relevance. It may be hypothesized that, like the anti-
antibodies, some cytosolic factor binds to the
subunit and disrupts
its interactions with the enzyme. Mutational analysis of the C-terminal
10 amino acids that comprise the epitope reactive with anti-
may
provide information on specific residues involved in
-
interactions.
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ACKNOWLEDGEMENTS |
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We thank Drs. Robert W. Mercer, Washington
University, and David C. Clarke, University of Toronto, for invaluable
suggestions and Mrs. Ania Wilczynska for technical assistance. The 6H
antibody was a generous gift from Dr. Michael Caplan, Yale University. We also acknowledge the Alberta Peptide Institute for synthesis of the
peptide used in generating antiserum C33.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada (MT-3876 to R. B.), the Quebec Heart and Stroke Foundation (to R. B.), and the Weizmann Institute Renal Research Fund (to S. J. D. K.).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.
The necleotide sequence reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number AF129400.
§ Recipient of a predoctoral scholarship from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
To whom correspondence should be addressed: Montreal General
Hospital, 1650 Cedar Ave., Rm. L11.132, Montreal, Quebec H3G 1A4,
Canada. Tel.: 514-937-6011, Ext. 4501; Fax: 514-934-8332; E-mail:
mirb{at}musica.mcgill.ca.
2 A. Therien, R. Daneman, and R. Blostein, unpublished observations.
3 R. W. Mercer, personal communication.
4 A. Shainskaya and S. J. D. Karlish, unpublished observations.
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ABBREVIATIONS |
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The abbreviation used is: 5'-RACE, 5'-rapid amplification of cDNA ends.
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