Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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ABSTRACT |
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We reported the identification of three outwardly rectified
Cl channel (ORCC) candidate
proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have
raised polyclonal antibodies against these isolated proteins.
Incorporation into planar lipid bilayers of material partly purified
from bovine tracheal apical membranes with one of these antibodies as a
ligand (anti-p115) resulted in the incorporation of an ORCC identical
in biophysical characteristics to one we previously described. We
developed a new purification procedure to increase the yield and purity
of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination of
anion- and cation-exchange chromatography followed by
immunopurification. By use of this purification scheme, 7 µg of the
115-kDa protein were purified from 20 mg of tracheal apical membrane
proteins. Incorporation of this highly purified material into planar
lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 ± 9 pS in symmetrical
Cl
solutions, halide
selectivity sequence of I
> Cl
> Br
, and lack of sensitivity
to protein kinase A, Ca2+, or
dithiothreitol. Using anti-G
i
antibodies to precipitate G
i
protein(s) from the partly purified preparations, we demonstrated that
the loss of rectification of the ORCC was due to uncoupling of
G
i protein(s) from the ORCC
protein and that the 115-kDa polypeptide is an ORCC.
115-kilodalton protein; immunoaffinity; Gi proteins
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INTRODUCTION |
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MANY PHYSIOLOGICAL STUDIES have demonstrated the
presence of outwardly rectified
Cl channels (ORCCs) in a
variety of cells, including human and bovine airway cells, colonic
epithelial cells, pancreatic duct cells, sweat gland cells, and
lymphocytes (5, 9, 13, 17, 21, 38). The unique biophysical properties
of ORCCs recorded using the patch-clamp technique or in planar lipid
bilayers are a nonlinear current-voltage
(I-V) relationship with a 30-pS
single channel conductance at hyperpolarizing voltages and an 80- to
90-pS conductance at depolarizing voltages (1, 4, 6, 8, 18, 21, 22), a
halide permeability sequence of
I
> Cl
> Br
(28), and activation by
protein kinase A (PKA) and protein kinase C (12, 21, 22, 40). PKA
activation of an ORCC is absent in epithelia lacking functional cystic
fibrosis transmembrane conductance regulator (CFTR) and can be restored
by transfecting wild-type CFTR (9). A requirement for the presence of
functional CFTR for PKA-dependent activation of the ORCC was also
confirmed in planar lipid bilayer studies (21, 22). A G protein,
G
i-2, inhibits the ORCC, as
demonstrated by patch-clamp studies (36) and in planar lipid bilayers
(18). Furthermore, in planar lipid bilayers, treatment of the
incorporated ORCC with pertussis toxin (PTX) conferred a linearity to
the previously rectified I-V curve of
the ORCC (18). Some nonphysiological stimuli can also activate the
ORCC: exposing silent patches to depolarizing voltages greater than +50
mV (17) or to trypsin (40) will induce activation of previously
quiescent ORCCs.
ORCCs can be blocked by a wide variety of different compounds, including DIDS, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, calixarenes, carboxylic acid, and indanyloxyl alkanoic acid derivatives (14-16, 37). Arachidonic acid and leukotrienes also can cause blockade of open channel currents (2, 37).
Despite the fairly detailed information concerning the
electrophysiological characteristics of this channel, the protein that forms the ORCC and the gene that codes for the protein are unknown. Our
laboratory has developed a protocol for the isolation of a 140-kDa
protein that forms a
Cl-selective,
Ca2+/calmodulin kinase II-, DIDS-,
and dithiothreitol-sensitive channel when incorporated into planar
lipid bilayers (7, 31, 32). Polyclonal antibodies have been raised
against a 38-kDa component of this homopolymeric protein. Using these
antibodies and immunopurification as the only step in the purification
scheme, we have isolated and reconstituted, in addition to the
Ca2+-activated
Cl
channel, an ORCC and
CFTR (18, 21, 22). Using in vitro phosphorylation of isolated proteins,
we have identified potential 115-, 85-, and 52-kDa ORCC candidate
proteins (21).
We report the generation of new antibodies raised against the
identified ORCC candidate proteins and an associated purification procedure designed to obtain a highly purified 115-kDa protein that
acts as an ORCC in bilayers. We also report the effect of Gi protein precipitation on the
rectification properties of ORCC.
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METHODS |
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Tracheal Apical Membrane Preparation
Apical membrane vesicles were prepared by differential centrifugation with use of a procedure first described by Langridge-Smith et al. (27) modified as previously described (30). Aliquots of vesicles (average protein concentration 5 mg/ml) were stored in liquid nitrogen until use.Peripheral proteins were extracted from native membrane vesicles by incubation of vesicles for 30 min in KCl buffer (100 mM KCl, 5 mM Tris-HEPES, and 0.5 mM MgCl2) titrated to pH 10.5 with 0.1 M NaOH. Alkaline-stripped vesicles were recovered by centrifugation at 100,000 g for 60 min.
Solubilization
These prewashed, peripheral protein-extracted apical membrane vesicles (200 µg) were solubilized with 200 µl of different concentrations of the detergents SDS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Triton X-100, and Nonidet NP-40 in the presence of KCl buffer (pH 7.4) and then centrifuged at 100,000 g for 60 min to remove unsolubilized material. Quantitation of solubilized protein was performed using the bicinchoninic acid protein assay. Solubilization of the 115-kDa protein was estimated using Western blotting and quantitative densitometry. Because 2% CHAPS was capable of solubilizing 95% of the 115-kDa protein, this detergent was used in further purification steps.Protein Separation on DEAE-Cellufine Anion Exchanger
The CHAPS extract (4 ml, 2 mg/ml protein) was incubated with 10 ml of preequilibrated DEAE-Cellufine resin (KCl buffer containing 2% CHAPS) for 60 min with occasional shaking. The resin was washed with 10 ml of the same buffer to remove nonbound proteins. This nonbound fraction contains the 115-kDa protein and was used in the next step of purification.Protein Separation on CM-Cellufine Cation Exchanger
The nonbound fraction from the previous anion-exchange separation step was combined with 10 ml of a CM-Cellufine resin equilibrated with CM buffer (MES-Tris + 2% CHAPS, pH 6.0) and incubated for 60 min with occasional shaking. The resin was washed with 10 ml of the same buffer to remove nonbound proteins and then eluted stepwise with buffers of higher pH as follows: 6.2, 6.5, 7.5, 7.8, and 8.5. The pH 6.2 and 6.5 fractions contained the 115-kDa protein and were combined and used in the next step of immunopurification.Immunopurification of Cl Channel
Proteins With Anti-p115 Antibodies
Highly purified preparation. A polyclonal rabbit antibody (anti-p115) generated against purified 115-kDa protein [isolated using anti-p38 antibodies (6, 26)] was covalently linked to a hydrazide-activated gel (Carbolink). Purified immune IgG was oxidized by metaperiodate (0.02 M) in the dark at room temperature for 1 h and coupled to the beads by incubation overnight at room temperature to reach maximum binding capacity (4-5 mg IgG/ml gel). The same amount of preimmune IgG was covalently linked to hydrazide-activated gel, and nonimmune beads were used as a control for the specificity of immunopurification. Before use, the Carbolink beads were washed extensively with MES-Tris buffer + 2% CHAPS, pH 7.4. To obtain the highly purified preparation, the pH 6.2 and 6.5 fractions eluted from the cation-exchange column were combined; pH was adjusted to 7.4, and the sample was incubated with the antibody-linked Carbolink beads for 1 h to allow binding of the protein to the antibody. The column was then extensively washed with MES-Tris buffer before elution with 100 mM glycine, pH 3. Successive 2-ml fractions were collected and immediately neutralized with 100 µl of 1.5 M Tris. The first two fractions that contained the most protein were pooled and concentrated to 100 µl and used for reconstitution in liposomes or for further biochemical characterization.
Partly purified preparation. To obtain the partly purified preparation, apical membrane vesicles were solubilized with 0.8% Triton X-100, and the cation- and anion-exchange steps were omitted, leaving immunopurification as the only step of purification after solubilization of integral membrane proteins.
Partly purified preparation after immunoprecipitation of
Gi protein(s).
Immunoprecipitation of G
i
protein(s) from partly purified ORCC material was performed using
polyclonal antipeptide antibodies (Calbiochem). These antibodies were
raised against a COOH-terminal decapeptide found in
G
i-1 and
G
i-2 proteins. The
immunopurified fractions containing partly purified 115-kDa protein (20 µg) were concentrated to a volume of 200 µl, and radioimmune
precipitation buffer (RIPA) was added (50 µl of 5× RIPA) to
ensure dissociation of G
i
protein(s) from the ORCC protein. RIPA buffer was also chosen on the
basis of previous experience that immunoprecipitation of CFTR in this
buffer did not affect biophysical properties of ORCC (21).
Anti-G
i-2 antibodies (5 µl)
were added and incubated at 4°C for 1 h. Then 20 µl of protein
A-Sepharose were added and incubated for 1 h at 4°C. After
centrifugation to pellet protein A, the supernatant was collected and
reconstituted as previously described (6) for bilayer studies. The
precipitated G
i protein(s) was
eluted from the protein A-Sepharose with use of 0.1 M glycine buffer,
pH 2.8, and used for coreconstitution experiments with highly purified
115-kDa protein.
PAGE and Western Blots
Protein separation on polyacrylamide gels was performed using the method of Laemmli (26). Proteins were transferred to polyvinylidine difluoride membranes by applying 100 V for 1 h or 30 V overnight. Blots were washed with TBS buffer (20 mM Tris, pH 7.5, and 500 mM NaCl) for 10 min and then incubated with 5% milk in TBS for at least 30 min. Primary antibodies (dilution 1:100 for anti-p115 and 1:500 for anti-GProtein Iodination
Protein iodination was carried out using the chloramine-T procedure (28). Highly or partly purified ClPlanar Lipid Bilayer Experiments
Immunopurified ClPlanar lipid bilayers, composed of 25 mg/ml of 2:1 (wt/wt)
phosphatidylethanolamine-phosphatidylserine, were painted with a
fire-polished glass capillary over a 200-µm hole drilled in a
polystyrene chamber, as described previously (19). Bilayer formation
was monitored by the increase in membrane capacitance to a final value
of 200-300 pF. Artificial liposomes containing Cl channel proteins were
incorporated into bilayers bathed with symmetrical solutions of 100 mM
KCl and 10 mM MOPS adjusted to pH 7.4. In some experiments, KCl was
substituted with 100 mM choline chloride or
N-methyl-D-glucamine
chloride. The observation of a stepwise increase in current was taken
as an indication of channel incorporation into the lipid bilayer.
Current measurements were performed with a high-gain amplifier circuit
based on a design previously described (18). Steady-state single
channel I-V curves were measured after
channel incorporation by applying a known voltage and measuring
individual channel current.
Production of Specific Antibodies for Immunopurification of ORCC
Using in vitro phosphorylation of immunopurified material isolated using anti-p38 antibodies, our laboratory has identified three ORCC candidate proteins (21). Repetitive immunopurification using anti-p38 antibodies was performed to isolate a sufficient amount of identified proteins for rabbit immunization. Immunopurified proteins were precipitated (methanol-chloroform), dissolved in 40 µl of sample buffer, and separated on gel under nonreducing conditions. The separated proteins were blotted onto nitrocellulose paper and stained by Ponceau S. The appropriate protein bands were cut from the nitrocellulose paper, dried in air, and dissolved in 200 µl of DMSO. DMSO-solubilized paper was mixed with an equal volume of complete Freund's adjuvant for the first injection or with incomplete adjuvant for subsequent injections. Approximately 6-8 µg of each protein were injected over a period of 3 mo (6 times at 2-wk intervals). After the last injection the rabbit was bled from an ear vein, and the blood was collected and left to coagulate at room temperature. After coagulation, blood was spun at 10,000 g for 20 min, and the clear serum was divided among vials and lyophilized. IgG purification from serum was performed using immobilized protein A-agarose, as previously described (21). ![]() |
RESULTS |
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Western Blot Characterization of New Antibodies (Anti-p115 and Anti-p52)
Immune serums were first tested for specificity on blots of total proteins extracted from bovine tracheal apical membranes (Fig. 1). Tracheal apical membranes were solubilized using 0.8% Triton X-100 in the presence of KCl buffer. The extracts of 50 µg of proteins were mixed with sample buffer, separated on a 10% gel, and immunoblotted. Under these conditions, immune IgGs (anti-p115 and anti-p52) reacted strongly with the original antigens (Fig. 1, A and B, lane 1). Preimmune IgGs showed no reactivity to any of the blotted proteins (Fig. 1, A and B, lane 2).
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These antibodies were subsequently used as ligands for immunopurification. Immunopurification was a single step of purification after solubilization of integral membrane proteins in these experiments. Immunopurified proteins were reconstituted and incorporated in planar lipid bilayers to test for channel activity. Only incorporation of immunopurified material obtained using the anti-p115 antibody resulted in ORCC activity.
Reconstitution of Partly Purified Material Obtained Using the Anti-p115 Antibody as a Ligand for Purification
Iodination of partly purified proteins isolated using the anti-p115 antibody as a ligand revealed the presence of polypeptides at 115, 40, and 31 kDa (Fig. 2). Reconstitution and incorporation of this partly purified material into planar lipid bilayers resulted in channel activity. Figure 3 shows representative traces of 11 recordings made with 3 individual preparations revealing a channel that exhibited marked rectification in symmetrical Cl
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Development of a Purification Scheme to Increase the Purity and Yield of the 115-kDa Protein
Since we have identified the 115-kDa protein as an ORCC candidate, we have developed a new purification procedure to increase the yield and purity of this polypeptide. To optimize the solubilization of the 115-kDa protein, several detergents were tested for their ability to solubilize integral membrane proteins as well as their ability to solubilize the 115-kDa protein (Table 1). Solubilization of membrane proteins was determined by the bicinchoninic acid protein assay. The amount of integral membrane protein solubilized with 5% SDS was considered as 100% solubilization, and solubilization with other tested detergents was expressed as a percentage of the solubilization achieved with 5% SDS. Solubilization with 5% SDS was also used as a reference for solubilization of the 115-kDa protein (Table 1). Quantitation of solubilized 115-kDa protein was performed using densitometry scanning of immunoblots of membrane extracts obtained with different detergents (Fig. 4). Inasmuch as we needed to preserve a functional channel protein through the last stage of purification to test for channel activity, only mild nondenaturating detergents were employed (CHAPS, Triton X-100, and Nonidet NP-40). The lowest concentration tested for any of these detergents was equal to the critical micellar concentration. To obtain optimal solubilization of the 115-kDa protein, higher concentrations of detergents were also tested (Table 1). Of the detergents screened, 2% CHAPS was the most effective in solubilizing the 115-kDa protein. CHAPS was capable of solubilizing 90 ± 6% of the 115-kDa protein from stripped tracheal membranes, whereas it solubilized only 73 ± 7% of integral membrane proteins. On the other hand, 0.8% Triton X-100 was very effective in solubilizing integral apical membrane proteins (80 ± 8%) but not so effective in solubilizing the 115-kDa protein (60 ± 7%). CHAPS is a mild zwitterionic detergent that contains head groups with positive and negative charges. This class of detergents is more efficient than nonionic detergents in overcoming protein-protein interactions while causing less protein denaturation than ionic detergents. CHAPS also does not interfere with ion-exchange chromatography and was included in all buffers in the subsequent purification step.
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Several chromatographic methods were examined for their ability to purify the 115-kDa protein. The purification scheme that yielded the best results in terms of overall yield and purity was a combination of anion- and cation-exchange chromatography followed by immunopurification. First, the separation profile of total solubilized membrane proteins on the anionic exchanger DEAE-Cellufine was tested. The CHAPS-solubilized extract in the presence of 100 mM KCl buffer was applied to the DEAE column, and then elution was carried out using a stepwise gradient of KCl (0.1-0.5 M KCl). Five 10-ml fractions were collected and analyzed by Western blot. Only the first, nonbound fraction contained the 115-kDa protein (Fig. 5, lane 2). As expected, the majority of tracheal membrane proteins (75%) were bound to the DEAE column (negatively charged) under these conditions. The nonbound fraction from the anion-exchange chromatography step was next applied to the cationic exchanger (CM-Cellufine), preequilibrated with MES-Tris buffer at pH 6, and incubated for 1 h. The 115-kDa protein was eluted in pH 6.2 and 6.5 fractions (Fig. 5, lane 3). The pH of these two fractions was adjusted to 7.4 before immunopurification. All buffers contained 2% CHAPS during purification. Protein concentration and yields of the 115-kDa protein during the purification procedure are shown in Table 2. The relative amount of the 115-kDa protein was estimated using densitometry scanning of immunoblots of all fractions during the purification procedure. Densitometry scanning of an amido black-stained blot of the highly purified 115-kDa protein was used to estimate the amount of 115-kDa protein after the final step of purification (Fig. 5C). Because the total amount of protein isolated from 20 ± 3 mg of starting tracheal apical membranes after the final immunopurification step was 8 ± 1 µg and 87% of this amount was 115-kDa protein (determined by densitometry scanning), we calculated that the amount of 115-kDa protein in this fraction was 7.0 ± 0.7 µg. Iodination of this highly purified material revealed a single band migrating at 115 kDa (Fig. 5D).
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Incorporation of Highly Purified 115-kDa Protein
A channel with a linear I-V relationship and unitary conductance of 87 ± 9 pS (n = 10) was recorded in planar lipid bilayers after incorporation of the proteoliposomes containing this highly purified 115-kDa protein (Fig. 6). The halide permeability of the recorded channel was I
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Analysis of Partly and Highly Purified ORCC Proteins for Presence
and Role of Gi Protein(s)
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DISCUSSION |
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We have described the successful production of new antibodies raised
against previously identified ORCC candidate proteins and a
purification procedure to obtain a high level of homogeneity for one of
these proteins (115-kDa protein). The main obstacles for purification
of ion channels from epithelia are the lack of specific ligands and the
small amount of channel proteins in epithelial cells. Using antibodies
previously raised (anti-p38) as a ligand for immunopurification, we
purified microgram amounts of the candidate ORCC proteins and raised
two new antibodies: anti-p52 and anti-p115. The specificities of the
new antibodies were confirmed by Western blotting. The antibodies
reacted strongly with the original antigens. Both antibodies were used
as ligands for immunopurification, but only incorporation of
immunopurified material obtained using the anti-p115 kDa antibody
resulted in ORCC activity when reconstituted into planar lipid
bilayers. The fact that anti-p38 antibodies were used to isolate the
115-kDa protein suggests a possible epitope similarity between the
previously isolated Ca2+-activated
Cl channel and the ORCC.
Four distinct structural classes of
Cl
channels have been
cloned. The first class includes the glycine and GABA receptors (11,
35), which belong to a superfamily of ligand-gated receptor channels.
The second class includes CFTR (33), which belongs to the gene family
of ATP-binding cassette transporters. The third class is the gene
family of ClC Cl
channels,
which include eight members so far (34). The fourth class is the gene
family of Ca2+-activated
Cl
channels (3). Although
they perform similar functions, there is no significant structural
homology among these different
Cl
channel classes.
Because we successfully raised specific antibodies (anti-p115), we were
able to develop a purification procedure to increase the yield and
purity of this 115-kDa protein. Quantitative immunoblotting was used to
determine optimal solubilization conditions for the 115-kDa
protein. Very good solubilization was achieved using the zwitterionic
detergent CHAPS. The same detergent was successfully used for the
solubilization of the ClC-0
Cl channel from
Torpedo californica electroplax (23).
Another advantage of using CHAPS over nonionic detergents is that CHAPS more efficiently overcomes protein-protein interaction. Detection and
estimation of the amount of the 115-kDa protein through the purification procedure were achieved by quantitative immunoblotting. The new purification procedure resulted in a high level of purification and a significant yield. A substantial amount of the 115-kDa protein (25 µg) was purified from 20 mg of tracheal apical membranes. Expressed as a percentage, our results suggest that isolated channel protein represents 1.2
of all membrane proteins. A similar
ratio of channel protein to total membrane proteins (1.3
) was
found for porin purified from bovine skeletal muscle (39). Recovery of
the 115-kDa protein through the purification procedure was 28 ± 8%, which was comparable to recovery of CFTR purified from Chinese
hamster ovary cells (29).
The highly purified protein was successfully reconstituted into planar
lipid bilayers. The incorporated channel had a linear I-V relationship as opposed to an
outwardly rectified I-V relationship, which was previously observed with the partly purified preparation. Precipitation of Gi protein
from the partly purified preparation resulted in a shift of the
outwardly rectified I-V curve to a linear profile. In addition, coreconstitution of
G
i protein (precipitated from
bovine tracheal epithelia) with highly purified 115-kDa protein induced
rectification of an otherwise linear anion channel, suggesting that
channel rectification was produced by interaction between G
i protein(s) and the channel.
We previously demonstrated that treatment of ORCC by PTX in planar
lipid bilayers (uncoupling a G
i
protein from ORCC) induced a shift from a rectified to a linear
I-V curve profile. Most studies of
Gi protein regulation of ion
channels have been performed using the patch-clamp technique. Many of
these studies demonstrated that G proteins can affect gating of ion
channels (20, 24, 25, 36), but it has not been shown that rectification
properties of a channel depend on interaction with G proteins. Using
three independent techniques to uncouple a
Gi protein from the channel
(precipitation with antibodies, PTX treatment, or purification of this
115-kDa protein), we have confirmed that uncoupling of the channel
protein from Gi protein induces a
shift from a rectified to a linear I-V
curve of the ORCC. This information will be valuable for the
interpretation of expression studies of this ORCC clone when available.
According to our results, it can be expected that rectification of the
I-V curve of an ORCC clone will depend
on the presence and coexpression of a
G
i protein in the expression
system.
In summary, we have developed a specific ligand (anti-p115) and
purification procedure for isolation of an ORCC from bovine tracheal
epithelia. The isolated 115-kDa protein was functionally reconstituted
into planar lipid bilayers. The reconstituted channel protein had a
rectified I-V relationship only in the
presence of a Gi protein.
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ACKNOWLEDGEMENTS |
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We thank Dr. Catherine Fuller for helpful discussions and for critiquing the manuscript several times during its preparation and Deborah Keeton for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48764 and by Cystic Fibrosis Foundation Fellowship F981 (B. Jovov). B. Jovov is also the recipient of a Parker B. Francis Foundation Fellowship.
Address for reprint requests: D. J. Benos, Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., 706 BHSB, Birmingham, AL 35294-0005.
Received 10 September 1997; accepted in final form 20 April 1998.
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