Vol. 182, No. 3, 1992 February 14, 1992
BIOCHEMICAL
CHARACTERIZATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1506-1513
OF IMMUNOREACTIVE IN HUMAN URINE
ENDOTHELIN
Koichi Am?, Tomoyuki Koshi’, Yuriko Ehara’, Toshiyuki Edano’, Masao Ohkuchi’, Mitsuteru Hirata’ and Tetsuro Okabe’ ‘Tokyo Research Laboratories, Kowa Co. Ltd., Tokyo 189, Japan ?he Third Department of Internal Medicine, Faculty of Medicine, Tokyo 113, Japan Received
January
University of Tokyo,
9, 1992
Summary: We developed three antibodies, specific and sensitive to endothelin-1 (ET-l), and established two sandwich and three competitive enzyme immunoassays (EL%). By using these EIAs, large immunoreactive ET (IR-ET) of molecular weight 10 k Da was identified as a main component of IR-ETs in human urine. This large IR-ET, which reacted with two antibodies specific for N-terminal region of ET-l but not with the antibody against Cterminal peptide of ET-l, was partially purified by six-step procedure and examined by Western blotting after SDS polyacrylamide gel electrophoresis. The large IR-ET was detected as a single band at molecular weight of 10 k Da both in reduced and non-reduced conditions. From these results, the large IR-ET was thought to consist of a single polypeptide chain and possess the steric restricted N-terminal region of ET-l. 0 1992 Academic Press, Inc. To investigate pathophysiological
role of endothelin (ET), which is the most potent
vasoconstrictor peptide consisted of 21-amino
acid residues (l), specific and sensitive
antibodies for ET were developed and several kinds of enzyme immunoassays @IAs) for evaluation ET concentration were established in many groups. Elevated levels of ET-l
in
body fluids of some patients have been reported from many institutes, however, the values of ET concentration in body fluids, especially in plasma, were different among institutes. These controversial results were thought to be attributed to the specificity of every antibody for ET and the extraction methods of immunoreactive ET (IR-ET) hand, some groups reported that IR-ET-like Abbreviations; ET, endothelin, B-GAL, B-D-gahtOSidase, horseradish peroxidase. 0006-291X/92 Copyright All rights
from plasma. On the other
substances (large H&ET), other than ET-1 and
IR-, immunoreactive-; ElA, enzyme immunoassay; HPLC, high-pressure liquid chromatography; HRP,
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1506
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3, 1992
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existed in plasma at molecular weights of 6 k Da (2, 3) or 11.6 k Da (4) by gel chromatography.
Although large IR-ET has not yet been well defined, it reacted
with ET specific antibodies and the dilution curve of large lR-ET was parallel to the ET-l standard curve (4).
Therefore, large IR-ET
results of EIAs for ET concentration.
was also one of the candidates affecting the
To determine the exact levels of ET-l,
it is urgent to
purify and identify these IR-ETs. We identified large IR-ET as a main component of IR-ETs
in human urine by our ET,
and here, we describe partial purification and several characteristics
MATERIALS
of this large IR-ET.
AND METHODS
Pep&&: ET-2, ET-3, porcine big ET-1[1-391 and Sarafotoxin S6b were obtained from Peptide Institute Inc. (Osaka). ET-l and following ET-l related peptides, ET-1[16-211, ET-1[4-lo], porcine big ET-1[22-391 and type B ET-l (ET-1 isomer linked with Cys’-Cys” and Cys3-Cysl’ bonds) and nicked ET-l (ET-1 cleaved at Lys9-Glu” bond by lysylendopeptidase) were obtained as previously described (5). ET-1[1-161, ET-l[l-191 and ET-1[1-201 (all containing Cys’-Cys” and Cys3-Cys” bonds) were prepared by cathepsin D and/or carboxypeptidase Y digestion and confirmed by amino acid analysis. . . -of ET-l and ET-1[16-211, conjugated to porcine thyroglobulin and emulsified with complete Freund’s adjuvant, was respectively administered subcutaneously in foot pad of rabbits and boosted 5 times at 2-week interval. One week after the last injection, anti-ET-1-IgG (ET-PA20) and anti-ET-l[lbal]-IgG (ET-PACl) were respectively obtained and purified with the use of Protein A-cellulofine column. A monoclonal antibody for ET-l (ET-MA51, IgG,) was prepared from BALBlc mice immunized ET-l conjugate (50 pg protein) as previously described (5). .. Qmpetrtrve EIG: Competitive EL4 was carried out as described previously (5). Briefly, ET-MA51, ET-PA20 or ET-PACl coated microtiter plates were incubated at 4°C for 16h with 100 pl of standard ET-l or samples in phosphate buffered saline (PBS) containing 0.05% (W/V) Tween 20 (PBS-T) and 100 yl of f3-D-galactosidase (g-GAL)-conjugated ET-l, diluted lOOO-fold with PBS-T. After washing each well with PBS-T, the bound enzyme activity was measured using 4-methylumbelliferyl-8-D-galactoside as a fluorescent substrate (5). &n&i&E& ET-MA51 or ET-PA20 coated microtiter plates were incubated at 4’C for 16h with standard ET-l (or sample, 200 pl) in PBS-T. After washing, 200 pl of B-GAL-conjugated ET-PACl solution diluted 500-fold with PBS-T, was added to the wells and the adsorbed conjugates were measured. Concentration Urine was collected from normal subjects and concentrated with a hollow fiber ultrafiltration system using HlP3-20 type cartridge (Amicon CO., USA). Gel The concentrated urine from 300 1, was precipitated with 50 % saturation of ammonium sulfate and collected by centrifugation (8,000 g x 30 min). The precipitate was dialyzed using SPECTRA/POR 3 against 20 mM Tris-HCl buffer, pH 7.4 (buffer A). The dialyzed solution was run on a Sephacryl S-200 HR column (2.6 x 94 cm) at 1.2 mUmin and eluted with buffer A containing 0.2 M NaCl. : The large IR-ET fractions obtained by gel exclusion chromatography were collected, loaded on a column of formyl- cellulofine coupled with 1507
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BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ET-PA20 (5 mg/ml resine, 2 x 6.4 cm), washed with PBS containing 1 M K!XN
until & became less than 0.1 and eluted with 1 M citrate solution. The IR-ET fractions were dialyzed against PBS, and was passed through a formyl-cellulofine coupled with normal rabbit y-globulin column (5 mg/ml resin, 2 x 6.4 cm) in order to exclude some proteins non-specifically bound to IgG. . . wchromatoera~hv All the passed-through &verse-wure solution described above was then acidified with 2 N of HCl to pH 4.0 and applied on an Amprep C, column (500 mg). After washing with 0.1 % trifluoroacetic acid (TFA), large IR-ET was eluted with 80 % acetonitrile containing 0.1 % TFA. The eluate, evaporated in vacua, was performed on a wondasphere C,, column (3.9 mm x 15 cm) and eluted with a linear gradient from 0 to 80 % acetonitrile in 0.1 % TFA at 0.4 ml/min. SDS-PAGE We&m.!&U&blottine: The samples were analyzed by the method of Laemmli et al. (6), in 20 % acrylamide containing 30 % ethylene glycol gel. The proteins on the SDS gel were transferred to PVDF membrane (7), and detected by chromogenic dye after treatment with ET-MA51 conjugated with horseradish peroxidase (HRP).
RESULTS The cross-reactivities of three antibodies with ET-related peptides were shown in Table 1. ET-MA51
and ET-PA20 were specific for the structure of ET-1[1-161
to type B or nicked ET-l. residues from C-terminal
and less reactive
They also reacted with the peptides lacking several amino acid of ET-l,
but only weakly with big ET-l.
ET-PACl
recognized
Table 1. Cross-reactivities of threecompetitiveEIAs for ET-relatedpeptides cross-reactivity(%) ET-relatedpeptides ET-MA51
ET-PA20
ET-PACl
ET-l ET-2 ET-3 porcinebig ET-1[1-391 SarafotoxinS6b
100 158 50.1 15.1 0.43
100 16.4 co.02 0.15 0
100 100 100 0.51 10.4
ET-1[1-161 (cyd-Cys’S, cys’-cysll) ET-l[l-19 (cys’-cys”, cys3-cys”) ET-1[1-201 (Cyd-cys”, cys3-Cys”)
100
100
0
100
100
0
100
100
0
ET-1[16-211 ET-1[4-lo]
type B ET-1[1-211 (cyd-cys”, cy&cys15) nickedET-1[1-9, lo-211 (cysl-cysls, cys3-Cys”) porcinebig ET-1[22-391
1508
0 0 9.5
0 0 5.6
100 0 100
0.1
0.31
100
0
0
0
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B l/100 l/20 l/5 l/200 l/)50 l/,10 y; . -.._-._. *-A__L_A
100
250 2 25
.:-. I
100
200 ET-1
300 ( rig/ml
400
I
0
500
1
0.1
)
ET-l
10 ( ngfml
100 )
Fig. 1. Dose response curves for ET-l in sandwich EIAs using ET-MA51 ( l ) and ETPA20 ( o ) (A) and competitive EIAs using ET-MA51 ( l ), ET-PA20 ( o ) and ET-PACl ( A ) (B) and serial difution curves of large IR-ET (broken line) (B).
ET-1[16-211
and was not affected with N-terminal
sequence or the steric structure, but C-
terminal Trp residue was very important. Using these antibodies, two sandwich (fig. 1A) and three competitive
EIAs (fig. 1B) were established.
The minimal detectable concentrations
were calculated 10 and 40 pg/ml, respectively, from the ET-1 standard curves.
Ten liters of human urine were concentrated with ultrafiltration
using YM-2
membrane,
and loaded on a Sephacryl S-100 HR column (1.6 x 98 cm). Fig. 2 illustrates those results
15
-10 F P L5 ":
0
50
100 Elution
150 Volume
200
( ml )
Fig. 2. Gel filtration profile of JR-ET in human urine measured by competitive EIA ( l ) and sandwich EIA ( o ) using ET-MA51. Arrows indicate the elution Positions of BSA (l), ovalbumin (2), chymotrypsinogen A (3), ribonuclease (4) and synthetic ET-1 (5). 1509
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?
1.5.
i ET-l
I I
,' t
1.0'
0.5' ,'
I'
0
3
O
lo Fraction
0
2o 3o 4o 5o) Number ( 5 ml/tube
4
'60
:
'40
;
-20
h '; ::
,,'
L
0
10
,'
.'
,'
,'
,'
20
Retention
,,'
30 Time
f 46
5b
( min
)
=r
Fip. 3. Affinity chromatography of large IR-ET on ET-PA20 coupled formyl-cellulofine. Fig. Reverse-phase HPLC profile of large IR-ET. of synthetic ET-l.
measured by sandwich
Arrow indicates the elution position
and competitive EIAs using ET-MA51.
Two major IR-ET
peaks
were observed and the former peak (fr. 120-150) was detected only by competitive EL4 and calculated 1Ok Da of molecular weight from marker proteins. this peak reacted with ET-MA51 ET-l,
but not with ET-PACl
position of synthetic ET-l
and ET-PA20
in parallel to standard solution of synthetic
(fig. 1B). The latter peak (fr. 185-200) was eluted at the same and reacted not only in competitive but also in sandwich
To investigate the former IR-ET (large IR-ET), further purification
Serially diluted fractions from
as described in “MATERIALS
300 1 of human urine was conducted to AND METHODS”.
More than 80 %
of large IR-ET was tiactionated by ammonium sulfate, and large IR-ET from other IR-ET ET-PA20
could be separated
by gel filtration as same as illustrated in fig. 2. Large IR-ET
an Amprep C, column.
The following
reverse-phase
was eluted earlier than authentic ET-l
was recovered from
HPLC yielded over 60 % and large
(fig. 4).
After six steps of purification
procedure, the sample solution still contained large amounts of brown-black 5 shows the results of Western blotting of the partially purified IR-ET. non-reduced
bound to
tightly enough to be retained on the ligand even after a washing with 1 M K!XN
and was eluted with 1 M citrate (fig. 3). About 40 % of large IR-ET
IR-ET
EIAs.
conditions, large IR-ET
detected as a single band, while ET-l
pigments.
Fig.
Both in reduced and
migrated to the same position of 10 k Da and was didn’t react with ET-MA51 1510
in reduced condition.
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,16949 14404 8159 6214
2512
Fig. 5. Western blotting of ET-l (A, B) and large IR-ET (C, D). Samples were applied without heating or treatment with 2-mercaptoethanol (A, C) and were heated for 3 min at 90°C in the presence of 5 % (V/V) 2-mercaptoethanol (B, D). Lane E contained molecular weight markers.
DISCUSSION It has been reported that the presence of IR-ETs, ET-1[1-391,
affect measurement of ET concentration
hemodialysis
patients (4) and rats (3).
We developed three ET-l competitive
EIAs.
which
specific antibodies, and established two sandwich
HPLC so that only ET-l
our EIAs, we detected large IR-ET
or big
in plasma of healthy human (2),
Ando et al. has reported that urinary IR-ET
itself on reverse-phase
differ from ET-l
and three
was co-eluted with ET-l
was present in human urine (8). By using
as a main component of IR-ETs
in human urine at the
molecular weight of 10 k Da by gel filtration chromatography (fig. 2). This discrepancy leads us to consider that our large IR-ET covalently bound to ET-l. filtration chromatography
would consist of ET-l
and a particular protein non-
We tried to separate the possible binding protein from ET-l.
was performed using several kinds of detergents and dissociation
reagents such as TRITON X-100, urea, guanidine hydrochloride, solution.
However,
Gel
large IR-ET
KSCN and others as eluting
couldn’t be divide into smaller components by any above 1511
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BIOPHYSICAL
RESEARCH
solution (data not shown). These results suggested that the large IR-ET two or more components but was a single chain polypeptide. ET-MA51
and ET-PA20,
The large IR-ET
standard
possessing two
and Cys3-Cys”) was necessary to be recognized by ET-MA51
and ET-PACl
that the large IR-ET
reacted with
(fig. 1B). From the results of cross-reactivities of three
antibodies with ET-related peptides (Table l), the structure of ET-1[1-161
and ET-PA20,
did not consist of
the dilution curves of which are parallel to the ET-l
curve, but not with the ET-PACl
disulfide bonds (Cysr-Cys”
COMMUNICATIONS
was specific for C-terminal region of ETs. These data suggest
would possess the similar structure to N-terminal
of ET-l
in the
molecule. To confirm these results, the large IR-ET
was further purified by gel filtration, affinity
chromatography using ET-PA20 and reverse-phase HPLC. Totsune et al. have reported that the C,* cartridge treatment could be used for the elimination
of large IR-ET
Tom plasma (4).
The low recovery of large IR-ET from the C, column in our experiment was consistent with their results, but in the following 2nd reverse-phase HPLC with the C,* column, the recovery became over 60 %.
The partially purified large IR-ET
was subjected to SDS-PAGE,
transferred to PVDF membrane and examined by Western blotting using HRP-conjugated ET-MA.51.
The large IR-ET
Interestingly,
the large IR-ET
migrated to a position of 10 k Da as a single band. reacted with ET-ml
both in reduced and non-reduced
conditions, while ET-1 reacted only in non-reduced condition (fig. 5). These results suggest that large IR-ET region to ET[l-163,
is a single chain polypeptide and possesses the similar steric restricted but is not affected by reducing agents.
Further studies are necessary to elucidate the complete structure of large IR-ET clarify its pathophysiological
role .
REFERENCES 1. Yanagisawa, M., Kurihara, H., Khnura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yaxaki, Y., Goto, K. and Masaki, T. (1988) Nature 332, 411-41s. 1512
and to
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2. Saito, Y., Nakao, K., Itoh, H., Yamada, T., Mukoyama, M., Arai, H., Hosoda, K., Shirakami, G., Suga, S., Jougasaki, M., Morichika, S. and Imura, H. (1989) Biochem.Biophys.Res.Commun. 161, 320-326. 3. Saito, Y., Nakao, K., Shirakami, G., Jougasaki, M., Yamada, T., Itoh, H., Mukoyama, M., Arai, H., Hosoda, K., Suga, S., Ogawa, Y. and Imura, H. (1989) Biochem.Biophys.Res.Commun. 163, 1512-1516. 4. Totsune, K., Mom-i, T., Takahashi, K., Ohneda, M., Sone, M., Saito, Y. and Yoshinaga, K. (1989) FEBS Lett. 249, 239-242. 5. Koshi, T., Torii, T., Arai, K., Edano, T., Hirata, M., Ohkuchi, M. and Okabe, T. (1991) Chem.Phann.Bull. 39, 1295-1297. 6. Laemmli, U. K (1970) Nature 227, 680-685. 7. Towbin, H., Staeheiin, T. and Gordon, J. (1979) Proc.Natl.Acad.Sci. U.S.A. 76, 4350-4354. 8. Ando, K., Hirata, Y., Takai, Y., Kawakami, M. and Marumo, F. (1991) Nephron 57, 36-39.
1513
BIOCHEMICAL
CHARACTERIZATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1506-1513
OF IMMUNOREACTIVE IN HUMAN URINE
ENDOTHELIN
Koichi Am?, Tomoyuki Koshi’, Yuriko Ehara’, Toshiyuki Edano’, Masao Ohkuchi’, Mitsuteru Hirata’ and Tetsuro Okabe’ ‘Tokyo Research Laboratories, Kowa Co. Ltd., Tokyo 189, Japan ?he Third Department of Internal Medicine, Faculty of Medicine, Tokyo 113, Japan Received
January
University of Tokyo,
9, 1992
Summary: We developed three antibodies, specific and sensitive to endothelin-1 (ET-l), and established two sandwich and three competitive enzyme immunoassays (EL%). By using these EIAs, large immunoreactive ET (IR-ET) of molecular weight 10 k Da was identified as a main component of IR-ETs in human urine. This large IR-ET, which reacted with two antibodies specific for N-terminal region of ET-l but not with the antibody against Cterminal peptide of ET-l, was partially purified by six-step procedure and examined by Western blotting after SDS polyacrylamide gel electrophoresis. The large IR-ET was detected as a single band at molecular weight of 10 k Da both in reduced and non-reduced conditions. From these results, the large IR-ET was thought to consist of a single polypeptide chain and possess the steric restricted N-terminal region of ET-l. 0 1992 Academic Press, Inc. To investigate pathophysiological
role of endothelin (ET), which is the most potent
vasoconstrictor peptide consisted of 21-amino
acid residues (l), specific and sensitive
antibodies for ET were developed and several kinds of enzyme immunoassays @IAs) for evaluation ET concentration were established in many groups. Elevated levels of ET-l
in
body fluids of some patients have been reported from many institutes, however, the values of ET concentration in body fluids, especially in plasma, were different among institutes. These controversial results were thought to be attributed to the specificity of every antibody for ET and the extraction methods of immunoreactive ET (IR-ET) hand, some groups reported that IR-ET-like Abbreviations; ET, endothelin, B-GAL, B-D-gahtOSidase, horseradish peroxidase. 0006-291X/92 Copyright All rights
from plasma. On the other
substances (large H&ET), other than ET-1 and
IR-, immunoreactive-; ElA, enzyme immunoassay; HPLC, high-pressure liquid chromatography; HRP,
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1506
Vol.
182,
No.
big ET-l, filtration
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
existed in plasma at molecular weights of 6 k Da (2, 3) or 11.6 k Da (4) by gel chromatography.
Although large IR-ET has not yet been well defined, it reacted
with ET specific antibodies and the dilution curve of large lR-ET was parallel to the ET-l standard curve (4).
Therefore, large IR-ET
results of EIAs for ET concentration.
was also one of the candidates affecting the
To determine the exact levels of ET-l,
it is urgent to
purify and identify these IR-ETs. We identified large IR-ET as a main component of IR-ETs
in human urine by our ET,
and here, we describe partial purification and several characteristics
MATERIALS
of this large IR-ET.
AND METHODS
Pep&&: ET-2, ET-3, porcine big ET-1[1-391 and Sarafotoxin S6b were obtained from Peptide Institute Inc. (Osaka). ET-l and following ET-l related peptides, ET-1[16-211, ET-1[4-lo], porcine big ET-1[22-391 and type B ET-l (ET-1 isomer linked with Cys’-Cys” and Cys3-Cysl’ bonds) and nicked ET-l (ET-1 cleaved at Lys9-Glu” bond by lysylendopeptidase) were obtained as previously described (5). ET-1[1-161, ET-l[l-191 and ET-1[1-201 (all containing Cys’-Cys” and Cys3-Cys” bonds) were prepared by cathepsin D and/or carboxypeptidase Y digestion and confirmed by amino acid analysis. . . -of ET-l and ET-1[16-211, conjugated to porcine thyroglobulin and emulsified with complete Freund’s adjuvant, was respectively administered subcutaneously in foot pad of rabbits and boosted 5 times at 2-week interval. One week after the last injection, anti-ET-1-IgG (ET-PA20) and anti-ET-l[lbal]-IgG (ET-PACl) were respectively obtained and purified with the use of Protein A-cellulofine column. A monoclonal antibody for ET-l (ET-MA51, IgG,) was prepared from BALBlc mice immunized ET-l conjugate (50 pg protein) as previously described (5). .. Qmpetrtrve EIG: Competitive EL4 was carried out as described previously (5). Briefly, ET-MA51, ET-PA20 or ET-PACl coated microtiter plates were incubated at 4°C for 16h with 100 pl of standard ET-l or samples in phosphate buffered saline (PBS) containing 0.05% (W/V) Tween 20 (PBS-T) and 100 yl of f3-D-galactosidase (g-GAL)-conjugated ET-l, diluted lOOO-fold with PBS-T. After washing each well with PBS-T, the bound enzyme activity was measured using 4-methylumbelliferyl-8-D-galactoside as a fluorescent substrate (5). &n&i&E& ET-MA51 or ET-PA20 coated microtiter plates were incubated at 4’C for 16h with standard ET-l (or sample, 200 pl) in PBS-T. After washing, 200 pl of B-GAL-conjugated ET-PACl solution diluted 500-fold with PBS-T, was added to the wells and the adsorbed conjugates were measured. Concentration Urine was collected from normal subjects and concentrated with a hollow fiber ultrafiltration system using HlP3-20 type cartridge (Amicon CO., USA). Gel The concentrated urine from 300 1, was precipitated with 50 % saturation of ammonium sulfate and collected by centrifugation (8,000 g x 30 min). The precipitate was dialyzed using SPECTRA/POR 3 against 20 mM Tris-HCl buffer, pH 7.4 (buffer A). The dialyzed solution was run on a Sephacryl S-200 HR column (2.6 x 94 cm) at 1.2 mUmin and eluted with buffer A containing 0.2 M NaCl. : The large IR-ET fractions obtained by gel exclusion chromatography were collected, loaded on a column of formyl- cellulofine coupled with 1507
Vol.
182, No. 3, 1992
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ET-PA20 (5 mg/ml resine, 2 x 6.4 cm), washed with PBS containing 1 M K!XN
until & became less than 0.1 and eluted with 1 M citrate solution. The IR-ET fractions were dialyzed against PBS, and was passed through a formyl-cellulofine coupled with normal rabbit y-globulin column (5 mg/ml resin, 2 x 6.4 cm) in order to exclude some proteins non-specifically bound to IgG. . . wchromatoera~hv All the passed-through &verse-wure solution described above was then acidified with 2 N of HCl to pH 4.0 and applied on an Amprep C, column (500 mg). After washing with 0.1 % trifluoroacetic acid (TFA), large IR-ET was eluted with 80 % acetonitrile containing 0.1 % TFA. The eluate, evaporated in vacua, was performed on a wondasphere C,, column (3.9 mm x 15 cm) and eluted with a linear gradient from 0 to 80 % acetonitrile in 0.1 % TFA at 0.4 ml/min. SDS-PAGE We&m.!&U&blottine: The samples were analyzed by the method of Laemmli et al. (6), in 20 % acrylamide containing 30 % ethylene glycol gel. The proteins on the SDS gel were transferred to PVDF membrane (7), and detected by chromogenic dye after treatment with ET-MA51 conjugated with horseradish peroxidase (HRP).
RESULTS The cross-reactivities of three antibodies with ET-related peptides were shown in Table 1. ET-MA51
and ET-PA20 were specific for the structure of ET-1[1-161
to type B or nicked ET-l. residues from C-terminal
and less reactive
They also reacted with the peptides lacking several amino acid of ET-l,
but only weakly with big ET-l.
ET-PACl
recognized
Table 1. Cross-reactivities of threecompetitiveEIAs for ET-relatedpeptides cross-reactivity(%) ET-relatedpeptides ET-MA51
ET-PA20
ET-PACl
ET-l ET-2 ET-3 porcinebig ET-1[1-391 SarafotoxinS6b
100 158 50.1 15.1 0.43
100 16.4 co.02 0.15 0
100 100 100 0.51 10.4
ET-1[1-161 (cyd-Cys’S, cys’-cysll) ET-l[l-19 (cys’-cys”, cys3-cys”) ET-1[1-201 (Cyd-cys”, cys3-Cys”)
100
100
0
100
100
0
100
100
0
ET-1[16-211 ET-1[4-lo]
type B ET-1[1-211 (cyd-cys”, cy&cys15) nickedET-1[1-9, lo-211 (cysl-cysls, cys3-Cys”) porcinebig ET-1[22-391
1508
0 0 9.5
0 0 5.6
100 0 100
0.1
0.31
100
0
0
0
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B l/100 l/20 l/5 l/200 l/)50 l/,10 y; . -.._-._. *-A__L_A
100
250 2 25
.:-. I
100
200 ET-1
300 ( rig/ml
400
I
0
500
1
0.1
)
ET-l
10 ( ngfml
100 )
Fig. 1. Dose response curves for ET-l in sandwich EIAs using ET-MA51 ( l ) and ETPA20 ( o ) (A) and competitive EIAs using ET-MA51 ( l ), ET-PA20 ( o ) and ET-PACl ( A ) (B) and serial difution curves of large IR-ET (broken line) (B).
ET-1[16-211
and was not affected with N-terminal
sequence or the steric structure, but C-
terminal Trp residue was very important. Using these antibodies, two sandwich (fig. 1A) and three competitive
EIAs (fig. 1B) were established.
The minimal detectable concentrations
were calculated 10 and 40 pg/ml, respectively, from the ET-1 standard curves.
Ten liters of human urine were concentrated with ultrafiltration
using YM-2
membrane,
and loaded on a Sephacryl S-100 HR column (1.6 x 98 cm). Fig. 2 illustrates those results
15
-10 F P L5 ":
0
50
100 Elution
150 Volume
200
( ml )
Fig. 2. Gel filtration profile of JR-ET in human urine measured by competitive EIA ( l ) and sandwich EIA ( o ) using ET-MA51. Arrows indicate the elution Positions of BSA (l), ovalbumin (2), chymotrypsinogen A (3), ribonuclease (4) and synthetic ET-1 (5). 1509
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?
1.5.
i ET-l
I I
,' t
1.0'
0.5' ,'
I'
0
3
O
lo Fraction
0
2o 3o 4o 5o) Number ( 5 ml/tube
4
'60
:
'40
;
-20
h '; ::
,,'
L
0
10
,'
.'
,'
,'
,'
20
Retention
,,'
30 Time
f 46
5b
( min
)
=r
Fip. 3. Affinity chromatography of large IR-ET on ET-PA20 coupled formyl-cellulofine. Fig. Reverse-phase HPLC profile of large IR-ET. of synthetic ET-l.
measured by sandwich
Arrow indicates the elution position
and competitive EIAs using ET-MA51.
Two major IR-ET
peaks
were observed and the former peak (fr. 120-150) was detected only by competitive EL4 and calculated 1Ok Da of molecular weight from marker proteins. this peak reacted with ET-MA51 ET-l,
but not with ET-PACl
position of synthetic ET-l
and ET-PA20
in parallel to standard solution of synthetic
(fig. 1B). The latter peak (fr. 185-200) was eluted at the same and reacted not only in competitive but also in sandwich
To investigate the former IR-ET (large IR-ET), further purification
Serially diluted fractions from
as described in “MATERIALS
300 1 of human urine was conducted to AND METHODS”.
More than 80 %
of large IR-ET was tiactionated by ammonium sulfate, and large IR-ET from other IR-ET ET-PA20
could be separated
by gel filtration as same as illustrated in fig. 2. Large IR-ET
an Amprep C, column.
The following
reverse-phase
was eluted earlier than authentic ET-l
was recovered from
HPLC yielded over 60 % and large
(fig. 4).
After six steps of purification
procedure, the sample solution still contained large amounts of brown-black 5 shows the results of Western blotting of the partially purified IR-ET. non-reduced
bound to
tightly enough to be retained on the ligand even after a washing with 1 M K!XN
and was eluted with 1 M citrate (fig. 3). About 40 % of large IR-ET
IR-ET
EIAs.
conditions, large IR-ET
detected as a single band, while ET-l
pigments.
Fig.
Both in reduced and
migrated to the same position of 10 k Da and was didn’t react with ET-MA51 1510
in reduced condition.
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,16949 14404 8159 6214
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Fig. 5. Western blotting of ET-l (A, B) and large IR-ET (C, D). Samples were applied without heating or treatment with 2-mercaptoethanol (A, C) and were heated for 3 min at 90°C in the presence of 5 % (V/V) 2-mercaptoethanol (B, D). Lane E contained molecular weight markers.
DISCUSSION It has been reported that the presence of IR-ETs, ET-1[1-391,
affect measurement of ET concentration
hemodialysis
patients (4) and rats (3).
We developed three ET-l competitive
EIAs.
which
specific antibodies, and established two sandwich
HPLC so that only ET-l
our EIAs, we detected large IR-ET
or big
in plasma of healthy human (2),
Ando et al. has reported that urinary IR-ET
itself on reverse-phase
differ from ET-l
and three
was co-eluted with ET-l
was present in human urine (8). By using
as a main component of IR-ETs
in human urine at the
molecular weight of 10 k Da by gel filtration chromatography (fig. 2). This discrepancy leads us to consider that our large IR-ET covalently bound to ET-l. filtration chromatography
would consist of ET-l
and a particular protein non-
We tried to separate the possible binding protein from ET-l.
was performed using several kinds of detergents and dissociation
reagents such as TRITON X-100, urea, guanidine hydrochloride, solution.
However,
Gel
large IR-ET
KSCN and others as eluting
couldn’t be divide into smaller components by any above 1511
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solution (data not shown). These results suggested that the large IR-ET two or more components but was a single chain polypeptide. ET-MA51
and ET-PA20,
The large IR-ET
standard
possessing two
and Cys3-Cys”) was necessary to be recognized by ET-MA51
and ET-PACl
that the large IR-ET
reacted with
(fig. 1B). From the results of cross-reactivities of three
antibodies with ET-related peptides (Table l), the structure of ET-1[1-161
and ET-PA20,
did not consist of
the dilution curves of which are parallel to the ET-l
curve, but not with the ET-PACl
disulfide bonds (Cysr-Cys”
COMMUNICATIONS
was specific for C-terminal region of ETs. These data suggest
would possess the similar structure to N-terminal
of ET-l
in the
molecule. To confirm these results, the large IR-ET
was further purified by gel filtration, affinity
chromatography using ET-PA20 and reverse-phase HPLC. Totsune et al. have reported that the C,* cartridge treatment could be used for the elimination
of large IR-ET
Tom plasma (4).
The low recovery of large IR-ET from the C, column in our experiment was consistent with their results, but in the following 2nd reverse-phase HPLC with the C,* column, the recovery became over 60 %.
The partially purified large IR-ET
was subjected to SDS-PAGE,
transferred to PVDF membrane and examined by Western blotting using HRP-conjugated ET-MA.51.
The large IR-ET
Interestingly,
the large IR-ET
migrated to a position of 10 k Da as a single band. reacted with ET-ml
both in reduced and non-reduced
conditions, while ET-1 reacted only in non-reduced condition (fig. 5). These results suggest that large IR-ET region to ET[l-163,
is a single chain polypeptide and possesses the similar steric restricted but is not affected by reducing agents.
Further studies are necessary to elucidate the complete structure of large IR-ET clarify its pathophysiological
role .
REFERENCES 1. Yanagisawa, M., Kurihara, H., Khnura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yaxaki, Y., Goto, K. and Masaki, T. (1988) Nature 332, 411-41s. 1512
and to
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2. Saito, Y., Nakao, K., Itoh, H., Yamada, T., Mukoyama, M., Arai, H., Hosoda, K., Shirakami, G., Suga, S., Jougasaki, M., Morichika, S. and Imura, H. (1989) Biochem.Biophys.Res.Commun. 161, 320-326. 3. Saito, Y., Nakao, K., Shirakami, G., Jougasaki, M., Yamada, T., Itoh, H., Mukoyama, M., Arai, H., Hosoda, K., Suga, S., Ogawa, Y. and Imura, H. (1989) Biochem.Biophys.Res.Commun. 163, 1512-1516. 4. Totsune, K., Mom-i, T., Takahashi, K., Ohneda, M., Sone, M., Saito, Y. and Yoshinaga, K. (1989) FEBS Lett. 249, 239-242. 5. Koshi, T., Torii, T., Arai, K., Edano, T., Hirata, M., Ohkuchi, M. and Okabe, T. (1991) Chem.Phann.Bull. 39, 1295-1297. 6. Laemmli, U. K (1970) Nature 227, 680-685. 7. Towbin, H., Staeheiin, T. and Gordon, J. (1979) Proc.Natl.Acad.Sci. U.S.A. 76, 4350-4354. 8. Ando, K., Hirata, Y., Takai, Y., Kawakami, M. and Marumo, F. (1991) Nephron 57, 36-39.
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