Department of Biochemistry, McGill University, Montreal, Canada H3G 1A4
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ABSTRACT |
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Tissue-distinct interactions of the
Na+-K+-ATPase
with Na+ and
K+, independent of
isoform-specific properties, were reported previously (A. G. Therien,
N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol.
Chem. 271: 7104-7112, 1996). In this paper, we
describe a detailed analysis of tissue-specific kinetics particularly
relevant to regulation of pump activity by intracellular
K+, namely
K+ inhibition at cytoplasmic
Na+ sites. Our results show that
the order of susceptibilities of 1 pumps of various rat tissues
to
K+/Na+
antagonism, represented by the ratio of the apparent affinity for
Na+ binding at cytoplasmic
activation sites in the absence of
K+ to the affinity constant for
K+ as a competitive inhibitor of
Na+ binding at cytoplasmic sites,
is red blood cell < axolemma
rat
1-transfected HeLa cells < small intestine < kidney < heart. In addition, we have
carried out an extensive analysis of the kinetics of
K+ binding and occlusion to the
cytoplasmic cation binding site and find that, for most tissues, there
is a relationship between the rate of
K+ binding/occlusion and the
apparent affinity for K+ as a
competitive inhibitor of Na+
activation, the order for both parameters being heart
kidney > small intestine
rat
1-transfected HeLa cells. The
notion that modulations in cytoplasmic
K+/Na+
antagonism are a potential mode of pump regulation is underscored by
evidence of its reversibility. Thus the relatively high
K+/Na+
antagonism characteristic of kidney pumps was reduced when rat kidney
microsomal membranes were fused into the dog red blood cell.
1-isoform; heart; kidney
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INTRODUCTION |
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THE NA+-k+-atpase, or sodium
pump, is a ubiquitous membrane protein complex that maintains the high
electrochemical gradient of Na+
and K+ across the plasma membrane
of animal cells (for reviews, see Refs. 13, 20, and 29). It comprises
two essential subunits, and
. The catalytic
-subunit
encompasses the sites of nucleotide and cation binding and undergoes
conformational transitions associated with the coupling of ATP
hydrolysis to the translocation of
Na+ and
K+. The
-subunit is required
for proper insertion and stability of the enzyme in the plasma membrane
and also has a role in modulating cation affinity (reviewed in Ref. 4).
Multiple isoforms of both the
(
1,
2,
3,
4)- and
(
1,
2,
3)-subunits are expressed in
a tissue- and development-specific manner (3, 19).
Although the basic function of the Na+-K+-ATPase is the maintenance of cation homeostasis, modification of its behavior in certain tissues may be critical to specialized functions such as Na+ reabsorption across epithelia, plasma K+ clearance by skeletal muscle, adjustment of the set point for Na+/Ca2+ exchange in the heart, and restoration of the electrochemical cation gradient after propagation of the nerve impulse. Relevant to such diversity of function in various tissues is an increasing body of evidence suggesting that the Na+-K+-ATPase is subject to complex short- and long-term regulation. In intact cells, sodium pump activity may be modulated by alterations in 1) intrinsic kinetic behavior, 2) cell surface expression, and 3) de novo pump synthesis (for reviews, see Refs. 2 and 8). Furthermore, the distinct properties of the different isoforms of the catalytic subunit and their putative distinct susceptibilities to regulatory processes comprise a diverse and elaborate set of modulatory mechanisms.
Although the nature of the -subunit isoform may be the primary
determinant of the intrinsic kinetic properties of the enzyme, other
cell-specific components may interact with and modulate kinetic
behavior. In an earlier study, we described tissue-specific differences
in the interactions of the enzyme with
Na+ and
K+ (28). A particular intriguing
finding, albeit rudimentary, was a notable difference in the effects of
K+ as a competitive inhibitor at
cytoplasmic Na+ activation sites
of pumps comprising either
1
1
or
3
1.
In this report, we describe a more extensive analysis of this
tissue-specific K+/Na+
antagonism and its mechanistic basis as it pertains to
1
1
pumps. Using the technique of fusing
1
1
pumps from one tissue (kidney medulla) into another (red blood cell),
we show that the tissue-specific effects are, in at least one instance,
subject to modification.
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MATERIALS AND METHODS |
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Cell culture and membrane
preparations. Rat
1-transfected HeLa cells were
grown and maintained in culture as described elsewhere (27). Membranes
from kidney, axolemma, and heart and
1-transfected HeLa cells and
red blood cells were prepared as described previously (27, 28).
Epithelial cells from small intestine were isolated by an adapted
method (18). Briefly, rat small intestines were sliced longitudinally
and washed with ice-cold 340 mM NaCl, and epithelial cells were
detached from the intestine by incubation in 240 mM NaCl containing 2.5 mM EDTA for 1 h at 4°C. After removal of the remaining intestine,
the detached cells and cellular debris were pelleted by centrifugation
for 15 min at 39,000 g. Membranes were
prepared from the pellet by the method used for kidney medulla. All
membrane preparations were stored at
70°C in a solution
containing 1 mM EDTA.
Enzyme assays. Before all experiments,
membranes were permeabilized by preincubation for 10 min at room
temperature in 15 mM Tris-Cl (pH 7.4) containing 1% BSA and 0.65 mg/ml
SDS, followed by dilution with 15 mM Tris-Cl (pH 7.4) containing 0.3%
BSA, essentially as described by Forbush (10). Such treatment was
previously shown to yield maximally permeabilized membranes, at least
for kidney (data not shown). Assays of
Na+-K+-ATPase
activity were carried out essentially as described previously (28),
except that 5 µM ouabain was included to inhibit the activity of
isoforms other than the rat 1.
Accordingly,
1-specific
activities were determined as the difference in ATP hydrolysis measured
in the presence of 5 µM ouabain and either 5 mM ouabain or 100 mM KCl
in the absence of NaCl, with no detectable differences between these
two baselines. Average activities of the membrane preparations of rat
kidney medulla, axolemma, heart,
1-transfected HeLa cell, red
blood cell, and small intestine membranes (µmol
Pi · mg
protein
1 · min
1)
were as follows: 2.8, 0.23, 0.14, 0.15, 0.05, and 0.5, respectively. For membrane preparations of mouse kidney
(whole), axolemma, and heart, the activities were: 0.84, 0.15, and
0.10, respectively. Assays of the
K+ dependence of
K+ occlusion
[E1 + K+
E2(K)], where E1 is the K+-free
enzyme and E2(K) is the occluded
enzyme, and the rate of deocclusion
[E2(K)
E1 + K+] were
also carried out as described elsewhere (6), with the following
modifications: 1) the final assay
volume was 100 µl and contained 5 µM ouabain,
2) ionic strength was kept constant at 4 mM with choline chloride during the preincubations with
K+, and
3) for the deocclusion assays,
enzyme was preequilibrated with 4 mM
K+, which was found to be
sufficient to form maximal E2(K)
for all tissues used.
Polyethylene glycol-mediated fusion of kidney microsomes to dog red blood cells. Fusion of rat kidney microsomes to dog red blood cells was carried out essentially as described by Munzer et al. (22) with minor modifications. Dog blood was collected into 1/10 volume of 0.1 M EDTA. The red blood cells were isolated by centrifugation (2 min at 500 g) and washed four times at 4°C with 10 volumes of wash buffer (140 mM NaCl, 10 mM KCl, 5 mM glucose, 68 mM sucrose, and 20 mM Tris-PO4, pH 7.4). The cells were then suspended and incubated in wash buffer for 1 h at 37°C, centrifuged, and washed one time at 4°C with wash buffer containing 1 mM adenosine and 0.5 mM adenine. One hundred fifty microliters of SHE solution (0.25 M sucrose, 0.03 M histidine, 1.0 mM EDTA-Tris, pH 7.4) or SHE containing 0.20-0.45 mg rat kidney microsomes was added to 1 ml of the packed red blood cells, and the cells were then incubated for 15 min at room temperature, followed by dropwise addition of 70% polyethylene glycol to a final concentration of 45%. The suspension was further incubated with gentle mixing for 45 s at room temperature, then 90 s at 37°C, followed by successive dilutions with 6, 14, and 26 ml of repletion buffer (wash buffer containing 1 mM adenosine, 0.5 mM adenine, and 2 mM MgCl2), with 30-s incubations at room temperature before each dilution. Cells were then incubated further for 1 h at 37°C, centrifuged for 5 min at 100 g, and washed four times at 4°C with 10 vol of wash buffer containing 2 mM MgCl2, by resuspension and centrifugation at 100 g. These last steps resulted in the isolation of cells free of unfused microsomes. Membranes from fused and mock-fused red blood cells were prepared by the same method as that used for rat red blood cells.
Analysis of kinetic data. Data for
Na+ activation of
Na+-K+-ATPase
activity and for K+ dependence of
K+ occlusion were expressed as
percentages of maximal activity or maximal occlusion, as determined by
extrapolation of the curves, using the Kaleidagraph computer program
(Synergy software) with the noninteractive model of cation binding
described by Garay and Garrahan (12)
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(1) |
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RESULTS |
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Previous studies of the Na+ and
K+ activation kinetics of the
Na+-K+-ATPase
have shown both isoform- and tissue-specific differences in apparent
affinities for Na+ and
K+ at activating cytoplasmic and
extracellular sites (17, 24, 28). Our earlier analysis provided
rudimentary evidence of tissue-specific interactions of
Na+ and
K+ at cytoplasmic sites that are
particularly relevant to the behavior of the pump in vivo under the
normal or resting steady-state condition of high
K+ and low
Na+ concentrations in the
cytosolic milieu (28). Thus the kidney 1
1
enzyme is notably more sensitive to
K+ inhibition at cytoplasmic
Na+ activation sites than pumps of
the same enzyme (rat
1
1)
of either rat
1-transfected
HeLa cells or axolemma, the latter two assayed in the presence of low
ouabain to inhibit activity due to ouabain-sensitive forms. The
comparative behavior of pumps of these and of other tissues is shown in
Fig. 1A.
The plots depict the activities of rat pumps as a function of
Na+ concentration under conditions
of relatively high (50 mM) K+
concentration. The behavior suggests that pumps of heart, kidney, and
intestine have lower apparent affinities for
Na+ compared with pumps of
1-transfected HeLa cells,
axolemma, and red blood cells. Figure
1B shows a similar pattern, albeit
with somewhat smaller differences, for pumps of another species
(mouse), namely heart, kidney, and axolemma.
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To gain insight into the mechanistic basis for these tissue-specific
differences, we carried out a series of analyses of the kinetic
behavior of 1 pumps of each
tissue, in which Na+ activation
profiles were determined as a function of
K+ concentration. As in our
previous study and based on the Albers-Post model with the assumption
that Na+ and
K+ bind randomly at three
equivalent cytoplasmic sites, the data were analyzed by the
relationship described by Garay and Garrahan (12), i.e.
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(2) |
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(3) |
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Analysis of
K+ interactions:
K+ occlusion and
the rate of the E2(K)
E1 + K+ reaction.
To gain more insight into the interactions of
K+ with cytoplasmic binding
site(s), a detailed analysis of the binding and occlusion of
K+ was carried out according to
the following simplified equilibrium relationship, which does not
distinguish the individual steps of sequential binding/occlusion of the
two K+
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(4) |
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Can tissue-specific differences in
K+/Na+
antagonism be attributed to distinct membrane environments?
To test whether susceptibility to high
K+/Na+
antagonism is reversible and related to the membrane environment of the
Na+-K+-ATPase,
pumps were transferred from the kidney to the red blood cell membrane
by the fusion procedure described earlier (21, 22). In those studies,
we showed that kidney microsomes can be functionally inserted in mature
red blood cells; those of the dog have the advantage of not containing
significant levels of endogenous
Na+-K+-ATPase.
In this study, we compared the Na+
activation profiles of fused and nonfused rat kidney pumps. We first
carried out fusions in the presence or absence of kidney microsomes.
Membranes were prepared, and the following three fusion conditions were
used for subsequent functional assays:
1) kidney microsome-fused red blood
cells, 2) kidney
microsome/mock-fused red blood cells (kidney microsomes added after a
mock fusion), and 3) mock-fused red
blood cells. Membranes from mock-fused red blood cells
(condition 3) contained no
ouabain-sensitive ATPase activity (data not shown). Figure
4 shows that kidney microsome/mock-fused red blood cells have a typically high
K'Na in the presence
of 100 mM KCl
[K'Na = 12.2 ± 2.4 mM, similar to values obtained with kidney pumps alone;
K'Na = 13.1 ± 1.7 (average of 12 experiments), data not shown]. However, membranes of kidney microsome-fused red blood cells showed a 1.9-fold decrease in K'Na
(6.3 ± 1.1 mM) toward that of 1 pumps of rat red blood cells,
axolemma, and
1-transfected HeLa cells.
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DISCUSSION |
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Several studies (17, 21) have clarified apparent anomalies noted in
earlier analyses of the
Na+-K+-ATPase
kinetics of tissues comprising different pump isoforms. In essence, it
is now clear that the distinct membrane environments of diverse tissues
are an important determinant of kinetic behavior. Notable cases in
point are the comparisons of 1
pumps of kidney with
3 pumps of
either the pineal gland (24) or axolemma (26). Whereas
3 pumps appeared to have a
higher apparent affinity for Na+
at cytoplasmic activation sites compared with
1 pumps of kidney, the order of
apparent affinities was reversed for
3 and
1 pumps transfected into HeLa
cells (17) or delivered from either rat axolemma or kidney into the dog
red blood cell (21).
A more detailed evaluation of the tissue- versus isoform-specific
behavior of pumps further underscored the importance of the membrane
environment as a determinant of pump behavior (28). In that study, it
became apparent also that there are notable tissue-specific differences
in the extent to which K+ behaves
as a competitive inhibitor at cytoplasmic
Na+ activation sites of pumps
comprising predominantly 1 or
3 catalytic isoforms. In fact,
the relative concentrations of Na+
and K+ under which
K+/Na+
antagonism is marked are those that prevail under steady-state physiological concentrations, and, as discussed below, this behavior may have important ramifications in relation to the regulation of
intracellular Na+.
In this study, the interaction of the enzyme with cytoplasmic
K+ was analyzed in terms of the
well-documented formation of
K+-occluded enzyme by the direct
binding of K+ to the
E1 conformation of the enzyme
(reviewed in Refs. 11 and 14). Based on the premise that the avidity of
K+ for cytoplasmic cation binding
sites should be evidenced in the rate of formation of the
K+-occluded enzyme
[E1 + K+ E1 · K+
E2(K); see
Eq. 4], the present analysis of
the K+ occlusion pathway of
several tissues that vary in
K+/Na+
antagonism (heart, kidney, small intestine, rat
1-transfected HeLa) shows an
intriguing inverse relationship between
ko, calculated from determinations of
Kocc and
kd as defined by
the simple equilibrium relationship in Eq. 4, and
KK measured under
steady-state conditions. Thus our data show a high rate of
K+ occlusion in the tissue (heart)
having a low KK
(high affinity for K+ as a
competitive inhibitor of Na+
binding), an intermediate rate in the tissue (kidney) with an intermediate KK,
and lower rates in tissues (HeLa, small intestine) with the highest
KK values. This
analysis was not extended to either axolemma or red blood cells, since
1) in axolemma, conditions for
measuring binding/occlusion and deocclusion of
K+ could be confounded by the high
proportion of the other (mainly
3) isoforms and
2) in the red blood cell, the
relatively high background activity and low specific activity of the
enzyme precludes accurate measurements of these parameters.
The correlation between the affinity constants
(KK) for
K+ as a competitive inhibitor of
Na+ binding (as determined by
plotting K'Na of
various tissues versus
[K+]; Table 1) and the
apparent rate constants for binding and occlusion of
K+
(ko; Table 2) in
pumps of kidney, heart,
1-transfected HeLa cells, and
small intestine membranes supports a model whereby K+/Na+
antagonism is related to the competing reactions represented by
1) binding/occlusion of
Na+ to form
E1P(Na) in the forward direction
and 2) binding/occlusion of
K+ to form
E2(K) in the backward reaction.
This result also supports a model of ion exchange whereby the
K+ release site and the
Na+ binding site are the same,
consistent with several studies showing that mutational or biochemical
alterations of residues deemed important for cation binding and
occlusion can affect both Na+ and
K+ interactions (23, 30).
Although Western blots of the various tissues showed a correlation
between 1 antibody reactivity
and amount of
1 activity analyzed in each lane (data not shown), there remains the possibility that our analysis is confounded by the presence of other
ouabain-sensitive ATPases, such as the putative "nongastric"
H+-K+-ATPases,
namely the "colonic"
H+-K+-ATPase
found in the colon, kidney, and uterus of mammals (5), the
H+-K+-ATPase
found in amphibian bladder (16), and the gene encoding a human
K+-dependent ATPase (ATP1AL1)
first cloned from human skin (15). We consider this possibility
unlikely, since, in the rat, mRNA for the colonic ATPase was not
detected in brain or small intestine, and only trace amounts were
detected in kidney and heart by Northern blot analysis, whereas the
ATP1AL1 message is absent in all of these tissues (5).
To better understand the structural basis for the observed differences in K+/Na+ antagonism, we compared the Na+ activation profiles (at 100 mM K+) of kidney pumps fused into dog red blood cells with that of unfused kidney pumps in the presence of mock-fused dog red blood cell membranes. Kidney membranes and dog red blood cells were used since kidney microsomal membrane preparations are predominantly right-side-out and thus are conducive to efficient fusion, and dog red blood cells are devoid of endogenous Na+ pumps (see Ref. 22). Our results show a notable decrease in K'Na of kidney pumps fused into red blood cells, consistent with the conclusion that either components of the red blood cell membrane are interacting with the exogenous pumps or components of the kidney membrane are dissociating from these pumps, resulting in a decrease in their susceptibility to inhibition by K+. This effect requires intimate interactions between the pumps and the membrane environment since the mere presence of mock-fused red blood cell membranes in the assay medium had no effect on activity. Although our results suggest an effect of the membrane environment, we cannot rule out another possibility: since fusion requires a 1-h incubation of the fused cells at 37°C to ensure integration of the exogenous pumps, it is possible that the pumps are somehow altered during this period by a cytosolic factor present in the red blood cell, for example, through phosphorylation/dephosphorylation. In either case, these results show clearly that the high affinity for K+ acting as a competitive inhibitor of Na+ binding, which is characteristic of kidney pumps, is reversible and can be regulated by some cellular component(s). This type of reversible modulation of exogenous pumps by components of the red blood cell membrane is reminiscent of the observed regulation of pumps by the so-called Lp-antigen of low-K+ sheep red blood cells. Using the same fusion strategy, we showed that interaction of Lp antigen with exogenous kidney pumps conferred the distinctive K+ inhibition of pumps of genetically low-K+ sheep red blood cells (31).
It could be suggested that the membrane component that
modulates susceptibility of the renal enzyme to
K+/Na+
antagonism is the so-called -subunit, since this peptide has been
detected in this but not in other tissues (27). We consider this
possibility unlikely for the following reasons:
1)
-subunit protein was not
detected in heart (27) even though pumps of this tissue are even more
susceptible to
K+/Na+
antagonism than kidney pumps, and 2)
the fusion experiments would indicate that the
-subunit dissociates
from kidney pumps upon fusion into red blood cells, yet fused and
unfused kidney pumps were inhibited by anti-
antiserum (cf., Ref.
27) to a similar extent (experiments not shown).
Association of 1 pumps with
distinct
isoforms is also unlikely to be the basis for
tissue-specific differences in
K+/Na+
antagonism. We have already shown in a previous report (28) that the
1 isoform associates only with
the
1 isoform in kidney, HeLa,
and axolemma, and we have since determined that the
1, but not the
2, isoform is detected in
Western blots of heart and small intestine (data not shown). In
addition, we consider it unlikely that the
-subunit could dissociate
from
upon fusion of pumps into the red blood cell. We cannot rule
out, however, that distinct
isoforms are the basis for the high
Na+ affinity (low
KNa; see Table 1)
of pumps of red blood cells, since message for the
2 and
3, but not the
1, isoforms was detected
recently in human reticulocytes (25).
In previous studies of Na+-dependent Rb+ transport (21), we showed that kidney pumps have a higher affinity for cytoplasmic Na+ than axolemma pumps when fused into dog red blood cells. However, in a more recent paper (28), we showed that native kidney pumps have a lower affinity for Na+ than axolemma pumps in Na+-activated ATPase assays. We postulated that this discrepancy may be related to a lower K+/Na+ antagonism after fusion of the pumps. Here we show that this hypothesis is at least partly correct. Thus the high K+ inhibition characteristic of kidney pumps is reversible and related to the membrane environment of the pump.
Because the concentration of K+ in cells is generally high compared with that of Na+ (roughly 10-fold higher), modulation of K+/Na+ antagonism could be a physiologically important mechanism of pump regulation, especially in tissues in which the role of the pump is specialized, such as the heart, the kidney, and the small intestine. Although the sodium pump has a fundamental role as a "housekeeping" transporter responsible for maintaining Na+ and K+ homeostasis in the kidney and small intestine, it is also responsible for the absorption or reabsorption of Na+ and other solutes that are transported across the apical membrane by Na+-dependent transporters (for reviews, see Refs. 7 and 9). In the heart, the sodium pump is an indirect regulator of cardiac muscle contraction, as originally formulated by Baker et al. (1). Thus variations in K+/Na+ antagonism of heart enzyme could alter intracellular Ca2+ via Na+/Ca2+ exchange secondary to alterations in cytoplasmic Na+ concentration. It is likely that such a regulatory mechanism extends to species other than the rat, since similar results were obtained with the murine enzyme. The reversibility of K+/Na+ antagonism as evidenced in the experiments with kidney pumps fused into red blood cells underscores the potential importance of intracellular K+ as a regulator of pump activity.
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ACKNOWLEDGEMENTS |
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We thank Drs. E. A. Jewell-Motz and J. B. Lingrel, University of
Cincinnati, for the generous gift of the rat
1-transfected HeLa cells and
Dr. Lawrence Joseph, McGill University, for help in the statistical
analysis of the data. The assistance of Ania Wilczynska and Richard
Daneman is gratefully acknowledged.
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FOOTNOTES |
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This work was supported by grants from the Medical Research Council of Canada (MT-3876) and the Quebec Heart and Stroke Foundation and by a predoctoral scholarship (to A. G. Therien) from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. Blostein, Montreal General Hospital Research Institute, 1650 Cedar Ave., Rm. L11.124, Montreal, Quebec, Canada H3G 1A4 (E-mail: MIRB{at}musica.mcgill.ca).
Received 3 May 1999; accepted in final form 14 July 1999.
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