816
Characterization of a Kininogenase from Rat Vascular
Tissue Resembling Tissue Kallikrein
Hector Nolly, A. Guillermo Scicli, Gloria Scicli, and Oscar A. Carretero
From the Hypertension Research Division, Henry Ford Hospital, Detroit, Michigan
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SUMMARY. A kininogenase resembling glandular kallikrein was partially purified from vascular
tissue and characterized. Saline-perfused rat tail arteries and veins were homogenized in 0.25 M
sucrose containing 10 ITIM Tris-HCl (pH 7.4). The homogenate was centrifuged at 105,000 g for
60 minutes, and a vascular kininogenase was purified from the supernatant by chromatofocusing,
affinity chromatography on immobilized antibodies against rat urinary kallikrein, and gel filtration
on Sephadex G-100. The inhibitory effects of antibodies against rat urinary kallikrein were tested
with equivalent kinin-forming concentrations of rat urinary kallikrein and vascular kininogenase.
Kininogenase activities of both enzymes were similarly inhibited by both polydonal and monoclonal antibodies. Aprotinin (1,000 JQU) completely inhibited vascular kininogenase activity,
while soybean trypsin inhibitor (100 /ig) did not modify its kinin-forming activity. Vascular
kininogenase and rat urinary kallikrein had the same elution volume when chromatographed on
a Sephadex G-100 column, and had similar mobilities in 10% polyacrylamide gel electrophoresis.
Kinins released by vascular kininogenase were identified as bradykinin by reverse-phase high
performance liquid chromatography. Rat vascular kininogenase appears to be similar to glandular
kallikrein. Kinins released locally by vascular kininogenase may contribute to the regulation of
vascular tone. {Circ Res 56: 816-821, 1985)
KALLIKREINS are enzymes that generate kinins
from protein substrates called kininogens (Webster,
1970). The ubiquity of these enzymes in different
tissues, the potent vasodilator action of kinins, and
their stimulatory effect upon prostaglandin synthesis in arteries are some of the data that support the
postulated role of the kallikrein-kinin system in the
regulation of vascular resistance. Tissue kallikreins
have recently been found in brain, thyroid gland,
intestine, stomach, and plasma (Rabito et al., 1980;
Lawton et al., 1981; Uetsuju et al., 1982; Uchida et
al., 1982; Chao et al., 1983; Schachter et al., 1983).
One of us has previously reported the presence of a
kininogenase in rat mesenteric arteries (Nolly and
Lama, 1982). However, in this study, contamination
with pancreatic kallikrein could not be excluded,
since, in the rat, the pancreas is spread into part of
the mesenteric region, hi the present study, we have
determined that a kinin-forming enzyme different
from plasma kallikrein is also present in tail arteries
and veins of the rat. We have partially purified this
vascular kininogenase and defined some of its biochemical characteristics.
Methods
Reagents
The following materials were obtained from commercial
sources: molecular weight markers, polybuffer anion exchanger (PBE 94), and polybuffer 74 (Pharmacia Fine
Chemicals); captopril (Squibb); polyacrylamide (Eastman
Kodak); all other reagents used were analytical grade.
Aprotinin was a kind gift from G. Haberland, (A.G. Bayer,
W. Germany).
Preparation of Tissue Extracts
One hundred male and female Sprague-Dawley rats
(400-500 g) were used. After an animal was killed by
decapitation, the ventral artery and veins of the tail were
exposed proximally through small incisions and catheterized with P.E. 50 polyethylene tubing (Friedman et al.,
1968). The tail was perfused through the artery with a
buffered saline solution (pH 7.4) for 10-20 minutes at a
flow rate of 1.0 ml/min until the effluent collected from
the veins appeared visually free of blood. Subsequently,
the vessels were removed, cleaned of any adherent fat
and connective tissue, and placed in Petri dishes containing a saline solution. The tail vascular bed was washed by
renewing the saline solution several times. Then, the
vessels were cut into 1-2 mm rings and kept frozen at
-70°C.
For the experiments, 600 mg of tail arteries, 800 mg of
tail veins, or 4,200 mg of pooled tail vessels were used.
The vessels were frozen and thawed four times. The tissue
was suspended in three volumes of 0.25 M sucrose (pH
7.0) and homogenized on ice for four 15-second intervals
with a Polytron homogenizer. The homogenate was centrifuged at 1,000 g for 10 minutes to eliminate debris. The
supernatant was further centrifuged at 105,000 g for 60
minutes in a L-5 Beckman ultracentrifuge. This supernatant was dialyzed overnight at 4°C against 0.01 M TrisHCl buffer (pH 7.0) for further purification. Kininogenase
activity was also found in the pellet dissolved in 0.1%
Triton X-100, but was much less than that present in the
supernatant. Further purification was done using the supernatant.
Nolly et a/./Kininogenase from Rat Vascular Tissue
Chromatofocusing on Polybuffer Anion Exchanger 94
(PBE94)
The vascular extract was applied to a polybuffer exchanger 94 column (20 X 1 cm) equilibrated with 0.025 M
imidazole-hydrochloride buffer (pH 7.4). A pH gradient
between 7.0 and 4.0 was obtained by using polybuffer
adjusted to pH 4.0. Proteins that were still bound to the
gel after pH 4.0 were eluted with 0.1 M sodium acetate
buffer, pH 3.5, 1 M NaCl. During acid elution, the pH was
rapidly neutralized by collecting in equal volumes of 2 M
Tris-HCl, pH 8.5. The fractions obtained after the sodium
acetate elution were pooled in three major fractions of 10
ml each. The fractions were dialyzed against distilled
water, concentrated by evaporating under a nitrogen
stream at 37°C, and frozen at -70°C until assayed for
kininogenase activity.
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Affinity Chromatography on Immobilized Kallikrein
Antibody
The 7-globulin fraction of the rabbit antiserum against
rat kallikrein was purified by precipitation with 40% ammonium sulfate. The kallikrein antibodies of this fraction
were further purified by adsorption to, and elution from,
a rat glandular kallikrein CH-sepharose column. For this,
glandular kallikrein was purified from rat submandibular
gland by chromatography on DEAE-Sephadex, adsorption
to and elution from a aprotinin-sepharose gel, isoelectric
focusing/ and gel filtration on high performance liquid
chromatography (HPLC) with a TSK-250 column. One
microgram of the purified enzyme released 1.1 ng of kinin
per minute when incubated with semi-purified dog kininogen (Carretero et al., 1976). In 12% slab polyacrylamide
gel electrophoresis, purified kallikrein (20 /ig) showed a
major and a minor band, both of which had kininogenase
activity. One milligram of purified rat submandibular
gland kallikrein was covalently bound to activated CHsepharose (Pharmacia) as indicated by the manufacturer.
The kallikrein CH-sepharose gel was equilibrated with
0.1 M Tris-HCl, 0.5 M sodium chloride, pH 8.0, and mixed
with the antikallikrein antibodies for 2 hours at room
temperature and then overnight at 4°C. After the gel was
washed to eliminate unbound material, the antikallikrein
antibodies were eluted with 0.1 M acetic acid, pH 2.9.
Purified kallikrein antibodies were coupled to activated
CH-sepharose (Pharmacia), as suggested by the manufacturer. The antikallikrein-sepharose gel was equilibrated
with 0.1 M sodium phosphate buffer, pH 7.4. The fraction
containing kininogenase after chromatofocusing activity
was dissolved in phosphate buffer and mixed with the
antikallikrein-sepharose gel for 2 hours at room temperature, followed by 24 hours at 4°C. Nonbound proteins
were separated by successive washings first with 0.1 M
sodium phosphate buffer (pH 7.4), and then with 0.1 M
sodium phosphate buffer (pH 6.0) containing 1 M NaCl.
The kininogenase activity was eluted with 0.1 M sodium
acetate buffer (pH 3.5) containing 1 M NaCl. Fractions (3
ml) were collected in equal volumes of 2 M Tris, pH 8.5.
The samples then were dialyzed against distilled water,
concentrated under a nitrogen stream, and frozen at
—70°C until assayed for kininogenase activity.
Gel Filtration on Sephadex G-100 and Molecular
Weight Determination
Two milliliters of the concentrated solution from the
immunoaffinity chromatography step were applied to a
Sephadex G-100 column (50 X 1 cm) equilibrated with
817
0.1 M phosphate buffer, pH 7.4. The column was eluted
at a flow rate of 20 ml/hr. Fractions (4 ml) were collected,
dialyzed against distilled water, and frozen at —70°C until
assayed. Standards used for molecular weight determination were ovalbumin, chymotrypsinogen, and ribonuclease.
Discontinuous Polyacrylamide Gel Electrophoresis
Disc gel electrophoresis was performed in 10% polyacrylamide gels as described by Nustad and Pierce (1974).
Each gel was cut into 2-mm slices, and each slice was
homogenized in the presence of 1 ml of 0.1 M sodium
phosphate buffer (pH 7.4), centrifuged at 4,000 g for 20
minutes, and the supernatant was saved for kininogenase
assay.
High Performance Liquid Chromatography of Kinins
Identification of kinins liberated during kininogenase
assay was carried out by comparing the elution volume of
kinins generated by the vascular homogenate with that of
synthetic bradykinin and lys-bradykinin during reversephase high performance liquid chromatography. Each
kinin solution was injected into a Cis Bondapack column
equilibrated with 10% acetonitrile in 0.05 M triethyl ammonium formate (TEAF), pH 4.4, and eluted with a linear
gradient (10% to 20% acetonitrile in 0.05 M triethyl ammonium formate, TEAF, pH 4.4) with a flow rate of
1 ml/min.
Kininogenase Assay
Kinin-generating activity in tissue extracts and fractions
were determined by incubation with partially purified dog
kininogen by a method identical to that previously described (Carretero et al., 1976), except that the buffer was
altered by the addition of captopril (10 jig/ml). Kinins
were measured by RIA (Carretero et al., 1976).
Inhibition Studies
The effect of soybean trypsin inhibitor (SBTT) and aprotinin on vascular kininogenase activity was examined.
SBTI was dissolved in 0.1 M Tris-HCl (5,000 Mg/ml) and
aprotinin was diluted with 0.1 M Tris-HCl (50,000
KIE/ml). The vascular kininogenase was preincubated
with 20 jil of the aforementioned inhibitors for 60 minutes
at 37°C. The final concentrations of inhibitors was 100 ng
of SBTI and 1,000 KIE of aprotinin per ml of sample. The
mixture was then incubated with kininogen for assay of
kininogenase as described. The 7-globulin fraction of the
rabbit antiserum against rat urinary kallikrein was diluted
in 0.1 M Tris-HCl buffer (pH 7.4) to a final concentration
of 20 mg/ml, and an aliquot containing 10 Mg proteins
TABLE 1
Kininogenase Activity in Tail Arteries and Tail Veina
Kininogenase
activity
% Inhibition
Blood vessel
pg/mg per hr
n
UKKAb
SBTI
Tail artery
Tail vein
605 ± 4 6
528 ± 23
6
8
72
87
3
5
Each experiment consisted of pooled arteries or veins (100 tng)
from three to five rats; n « number of experiments; UKKAb =
urinary kallikrein antibodies; SBTI ** soybean trypsin inhibitor
(100 Mg/ml); mean values ± SE. Kininogenase activity is expressed
per mg protein per hour of incubation.
Circulation Research/Vo/. 56, No. 6, June 1985
818
TABLE 2
Purification of Vascular Kininogenase from Rat Tail Vessels
Total
purification step
Total
Protein (mg) activity
1.68
2.12
1.92
1.60
1.80
1.08
0.028
0.022
Supernatant, 105,000 g
Chromatofocusing
Immunoaffinity
Gel filtration
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
was added to the samples of vascular kininogenase or
urinary kallikrein and preincubated for 60 minutes at
37°C. As control, nonimmunized rabbit serum was used
in the same concentration. After the preincubation period,
the mixture was incubated with kininogen for assay of
kininogenase activity. Monoclonal antibodies against rat
glandular kallikrein were kindly provided by Dr. R. SavoyMoore (Savoy-Moore et al., 1984). Subclones were propagated by intraperitoneal injection in mice. The ascites
fluid containing monoclonal antibodies against kallikrein
was used. As a control, we used normal mice serum diluted
1/400 with 0.1 M Tris-HCl, pH 7.4
Protein determination was done by the method of Bradford (1976), using bovine serum albumin as the standard.
Protein concentration in column effluents was measured
at 280 nm (A^)Rat urinary kallikrein was purified with a modification
of a previously described method (Oza et al., 1976). In
brief, proteins from rat urine obtained by ammonium
sulfate precipitation were chromatographed on a DEAE
A52 cellulose column. Fractions containing kininogenase
activity were adsorbed to and eluted from a aprotininCH-sepharose. Further purification was performed by size
exclusion in high performance liquid chromatography using a bio-sil TSK-250 column. Purified rat urinary kallikrein was homogeneous in 12% alkaline polyacrylamide
gel electrophoresis and released 1.2 ^g of kinin per
Purification factor
(Mg BK/mg
perhr)
Specific activity
(Mg BK/hr)
0.9
2.0
1
2.1
68.4
72.7
73.3
77.9
Yield
(%)
100
125
113
95
Mg protein per min when incubated with semipurified dog
kininogen.
Rat plasma kallikrein was prepared from blood collected
into 1/10 volumes of 0.1 M sodium citrate solution and
centrifuged at 1500 g for 30 minutes at 4°C. Acetone was
added to the plasma (17% vol/vol) for 18 hours at room
temperature. The acetone was evaporated under a nitrogen stream. After correction for water loss, the preparation
was kept at —20°C until needed.
Results
Table 1 shows the kininogenase activity of several
preparations of tail arteries and veins. The mean
values of kininogenase activity and the inhibition
profile with kallikrein antibodies and soybean-trypsin inhibitor were similar for both types of blood
vessel.
The results of the partial purification of rat vascular kininogenase are summarized in Table 2.
Chromatofocusing resulted in a 2-fold increase in
the specific activity, but total activity also increased
after this step. With immunoaffinity, the specific
activity increased up to 68-fold. After gel filtration,
a 78-fold purification with 95% recovery was obtained. On chromatofocusing, vascular kininogenase
and purified rat urinary kallikrein eluted together
when pH reached 3.5 (Fig. 1). This indicates that
their isoelectric point is below 4.0.
6
4
2 i
0 O
0.4 K"l
o
1
1
^
CO
CM
I?
r
6
<
4
'
2
40
60
80
Elutlwi Volumtlml)
IOO
FIGURE 1. Chromatofocusing on polybuffer anion exchanger 94 (PBE
94). Vascular kininogenase and urinary kallikrein were applied to a
column of PBE 94 (20 X 1 cm) equilibrated with 0.025 st imidazoleHCl buffer (pH 7.4). When the pH was 4.0, the vascular kininogenase
and urinary kallikrein were eluted with 0.1 M sodium acetate buffer,
1 M NaCl (pH 3.5). Fractions of 2 ml were collected over 2 ml of 2 M
Tris-HCl, pH 8.5. Open circles and triangles indicates inhibition of
the kininogenase activity by incubation with kallikrein antibodies.
I 0.3
o
~
<
\
\
\
\
i
1
0.2 1
1 1
I
\
\
V
fl
200
1 with
^KKAb
\
•100
O.I rr
1
• 300
\
•
^
V
•
J
5 3
1
•
i
i
10
IS
20
Elution Volume (ml)
pH7.4
pH6.0 '
pHJ.5
FIGURE 2. Affinity chromatography of vascular extract on antikallikrein-CH-sepharose. The gel was equilibrated with 0.1 M sodium
phosphate buffer (pH 7.4). Weakly bound proteins were eluted with
0.1 M sodium phosphate, 1 u NaCl (pH 6.0), and the vascular
kininogenase was eluted with 0.1 u sodium acetate buffer, 1 MNaCl
(pH 3.5). Open circles indicate inhibition of the kininogenase activity
by incubation with kallikrein antibodies.
819
Nolly et al. /Kininogenase from Rat Vascular Tissue
500 r Votculor
Klnlnogtnatt
320
240
160
400
Chymotryptinogen
300
j
' Vascular Kininogenoae
80
0
200
100
10
E
0
£ i
™
600
m
]i
500
5
400
J
300
32
36
40
c c
640
| ~
480
320
Chymotryptinoaen
OvaltMjmin
I
1
25
15
20
25
Urinary Kallikreln
160
0
5
with
KK-Ab
100
20
60
Urlnory
KolllKrtln
200
0
44
15
10
Gel Slice Number ( - 2mm each)
32
36
40
44
60
Elution Volume (ml)
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FIGURE 3. Gel filtration of the purified vascular and urinary kininogenasts on Sephadex C-100. The column was eluted at a flow rate of
20 ml/hr. Fractions of 4 ml were collected. Open circles and triangles
indicate inhibition of the kininogenase activity by incubation with
kallikrein antibodies.
On immunoaffinity chromatography, the kininogenase activity was eluted in a single peak with
0.1 M sodium acetate buffer (pH 3.5), 1 M NaCl (Fig.
2). Vascular and urinary kininogenases also eluted
in a single peak on gel filtration (Fig. 3). Kininogenase activity of both vascular and urinary kallikrein
was inhibited by incubation with kallikrein antibodies. The apparent molecular weight of vascular kininogenase as calculated from the elution volume on
the gel filtration column was 37,000 daltons (Fig. 4).
Mobility on disc polyacrylamide gel electrophoresis of both vascular kininogenase and urinary kallikrein was similar (Fig. 5).
The kininogenase activity of both vascular kininogenase and urinary kallikrein was completely inhibited (98 ± 2%) by aprotinin (not shown in the
figure), whereas it was resistant to soybean trypsin
inhibitor. In contrast, the kininogenase activity of
trypsin and plasma was strongly inhibited by soybean trypsin inhibitor (Fig. 6). Kininogenase activity
of both enzymes was inhibited to the same extent
Ovolbumln (mw 4 3 , 0 0 0 )
^Vascular Kininogenase (mw 37,000)
Chymotrypsinogen
(mw 25,000)
Rlbonucleoje
(mw 13,700)
100
Elution Volume (ml)
FICURE 4. Determination of the molecular weight of vascular kininogenase by gel filtration on a Sephadex G-100 column.
FIGURE 5. Alkaline disc gel electrophoresis of the purified vascular
kininogenase and of urinary kallikrein. Each slice (2 mm) was homogenized and cenlrifuged; the kininogenase activity was measured
in the supernatant.
by kallikrein antibodies. Nonimmunized rabbit
serum had a negligible effect.
Identification of the reaction products by reverse
phase HPLC showed that kinins generated by vascular kininogenase had the same elution volume as
synthetic bradykinin (Fig. 7).
Vascular
Kinlnoqenate
100
0
20
40
60
80
100
0
20
40
60
80
100
Incubated with polyclonal KK-Ab
Incubated with monoclonal KK-Ab
IT
Incubated with toybean-tryptin Inhlb.
FIGURE 6. Effect of kallikrein antibodies and soybean trypsin inhibitor
on kinin-generating activity of vascular kininogenase, urinary kallikrein, trypsin, and rat plasma (plasma kallikrein). The different
enzyme preparations were incubated for 60 minutes at 37°Q pH 8.5,
with kallikrein antibody, nonimmunized tabbit serum, or soybean
trypsin inhibitor, then kininogenase activity was measured. The
values represent the mean of at least three determinations.
Circulation Research/Vol. 56, No. 6, June 1985
820
Synthetic Kinin Standards
300
LYS-Bk
-Bradyklnlr
1)
200
100
Bk
'• J
Vascular Tissue
Kininog*flos* Products
dyk Inin
6 ?
300 -
&
200
100
1
'
A
20 22 24 26 28 30 32 34 36 38
Elution Volumt (ml)
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
FIGURE 7. Elution of synthetic kinin standards or kinin generated by
the vascular extract. The column was equilibrated and eluted at a
flow rate of 1.0 ml/min, as described in Methods. Kinin content of
the fractions was measured by R1A Upper injection of a mixture of
synthetic bradykinin (Bk) or Lys-bradykinin (LYS-Bk). Lower injection
of the kinin product of the vascular kininogenase.
Discussion
The present study demonstrates that a kininforming enzyme is present in rat vascular tissue.
Vascular kininogenase was purified by a series of
chromatographic steps with a very high yield. This
was unexpected, since the combination of these
chromatographic steps usually results in much lower
recoveries (Fiedler, 1979). Whereas we do not have
an explanation for this high yield, it is possible that
either activation of a proenzyme or separation from
inhibitors may have occurred during purification.
Chromatofocusing is likely to be an 'activating' step,
since it yielded 125% of the loaded activity. The
insensitivity of the vascular kininogenase to SBTI
readily distinguishes it from trypsin and plasma
kallikrein. On the other hand, rat vascular kininogenase appears to be very similar to rat glandular
kallikrein; both purified rat vascular kininogenase
and urinary kallikrein had similar molecular
weights, isoelectric points, electrophoretic mobilities,
and inhibition profiles. Only bradykinin was released by vascular kallikrein. This is not surprising,
since rat glandular kallikrein differ from similar
enzymes from other species in that they generate
bradykinin and not lys-bradykinin (Seki et al., 1972;
Alhenc-gelas et al., 1981; Yamada and Erdos, 1982).
The similar behavior of both enzymes when reacted with rabbit antiserum against rat urinary kallikrein strengthens the contention that they are very
similar if not identical. Vascular kininogenase and
urinary kallikrein were strongly bound to kallikrein
antiserum coupled to CH-sepharose, since they were
not released when the gel was washed with buffer,
pH 6.0, containing 1 M NaCl. The property of the
vascular kininogenase to bind strongly to immobilized kallikrein antiserum was used for its partial
purification from vascular tissue. The combination
of chromatofocusing, gel filtration, and immunoaffmity chromatography resulted in a 70-fold purification. Due to the small amount of material available, the analytical techniques usually used to determine purity were not performed.
We do not know at this time the precise localization of vascular kininogenase within the vascular
structures. Recently, it has been reported that mast
cells from humans and rats have a kallikrein-like
enzyme (Proud et al., 1982; Garret et al., 1984), and
it has been clearly demonstrated that these cells are
normally present around the arterioles, capillaries,
venules, and beneath the endothelium in man and
rats (McGovern, 1956; Robinson et al., 1978; Plonsky and Boyles, 1981).
In our experiments, the presence of mast cells
cannot be ruled out, but recent studies have demonstrated that the kininogenase present in human
mast cells is not immunologically cross-reactive with
glandular kallikrein (Proud et al., 1982). In addition,
the one present in rat mast cells is not inhibited by
aprotinin (Garret et al., 1982; Garret et al., 1984).
These two important differences should be taken
into consideration when comparing them with the
vascular kininogenase described here.
Although the precise cellular localization of vascular kininogenase is still unknown, it could be
speculated that the enzyme may play a role in
regulating local vascular resistance and, hence,
blood flow, either directly or through kinin release.
If the enzyme is located in the endothelial lining, it
may participate in the regulation of phospholipase
activity and local formation of arachidonic acid metabolites, either directly (Morita et al., 1984), or
through kinin formation (Whorton et al., 1982). If
the enzyme is located in smooth muscle, it may
participate directly in regulation of vascular tone,
and, if located in the adventitia, could have a role
in regulating vascular permeability. Furthermore, a
recent report (Cardin et al., 1984) suggests that low
density lipoproteins are a substrate for tissue kallikrein; thus, tissue kallikrein may participate in low
density lipoprotein metabolism within the vascular
wall.
In summary, a protease(s) which resembles glandular kallikrein is present in vessels isolated from
rat tails. This enzyme is present in an active form,
and its isoelectric point, molecular weight, and immunological characteristics are similar to those of
urinary kallikrein.
We would like to thank Pat Piejak for typing, and Dr. Robert
Murray for editing the manuscript.
This work was supported in part by National Institutes of Health
Grants HL 28981 and HL 15839
Dr. Nolly is an Established Investigator of the Argentine Council
of Research (CONICET). His present address is: Universidad National
Jolly et a/./Kininogenase from Rat Vascular Tissue
e Cuyo, School of Medicine, Department of Pathology, Mendoza,
rgentina.
Address for reprints: Oscar A. Carretero, M.D., Henry Ford Hosital, Hypertension Research Division, 2799 West Grand Boulevard,
etroit, Michigan 48202.
Received December 14, 1984; accepted for publication February
7, 1985.
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INDEX TERMS: Kininogenases • Kallikrien-kinin system • Arterial wall • Kinins and blood pressure • Serine protease
Characterization of a kininogenase from rat vascular tissue resembling tissue kallikrein.
H Nolly, A G Scicli, G Scicli and O A Carretero
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Circ Res. 1985;56:816-821
doi: 10.1161/01.RES.56.6.816
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