Clinical Science (1979) 57,345-350
Characteristics of a renin-binding substance for the
conversion of renin into a higher-molecular-weight form in
the dog
MINORU KAWAMURA, FUMIHIKO IKEMOTO, SUSUMU FUNAKAWA AND
KENJIRO YAMAMOTO
Department of Pharmacologv, Osaka City University Medical School, Osaka, Japan
(Received 8 February 1979; accepted 30 May 1979)
Summary
1. Renal cortical homogenates of the dog were
subjected to sieve separation, a Nucleopore Filter
being used to separate the renin granules.
2. The molecular weight of renin in the granules
was estimated to be about 40 OOO by gel filtration.
Renin was converted into a higher-molecularweight form (60 OOO) by mixing with cytosol in the
presence of sodium tetrathionate, a thiol inhibitor.
3. When cytosol was pretreated with acid (pH
3.0) or heating (lOOOC), the molecular-weight
conversion did not occur.
4. Cytosol was separated into three parts by gel
filtration. Fraction A included substances with a
molecular weight of over 47 0o0, fraction B from
47 OOO to 32 000,and fraction C from 32 OOO to
15 OOO. The mixture of renin in the granules with
fraction A and sodium tetrathionate resulted in the
formation of a higher-molecular-weight form of the
enzyme, but no change in molecular weight was
detected when renin was mixed with fractions B or
C and sodium tetrathionate.
Key words: acidification, cytosol, granule, highermolecular-weight form of renin, renin, reninbinding substance.
Introduction
Since the original observation by Boyd (1972) of a
higher-molecular-weight form of renin, there have
Correspondence: Dr Minoru Kawarnura, Departmenl
of Pharmacology, Osaka City University Medical
School, 1-4-54 Asahimachi, Abeno,Osaka 545, Japan.
345
been a number of reports of its existence in the
kidney and plasma in various species: rabbits
(Leckie, 1973; Leckie & McConell, 1975), rats
(Morris & Johnston, 1976), mice (Malling &
Poulsen, 1977), pigs (Boyd, 1974; Inagami &
Murakami, 1977; Inagami, Hirose, Murakami
& Matoba, 1977; Levine, Lentz, Kahn, Dorer
& Skeggs, 1978), dogs (Funakawa, Funae &
Yamamoto, 1978) and man (Day & Luetscher,
1974; Day, Luetscher & Gonzales, 1975; Weinberger, Wade, Aoi, Usa, Dentino, Luft & Grim,
1977; Slater & Haber, 1978). However, little is
known of the physiological role of the highermolecular-weight form of renin, nor of the mechanism and relevance of its conversion into a lowermolecular-weight form. Inagami et al. (1977)
reported that the higher-molecular-weight form of
renin was extractable from pig and rat kidney
homogenates whose protease activity was prevented by several inhibitors, and concluded that the
higher-molecular-weight form of renin in the kidney
was able to change to the lower-molecular-weight
form by interaction with a protease in the tissue.
Such a conclusion suggests that the highermolecular-weight form of renin is stored as a
precursor of the lower-molecular-weight form.
Recently, however, Funakawa et al. (1978)
found that the renin in isolated granules from dog
kidney was in the lower-molecular-weight form,
and capable of being converted into the highermolecular-weight form when mixed with the soluble
fraction of renal cortical homogenate in the
presence of protease inhibitors. This result clearly
indicated that stored renin was in the lower-
346
M . Kawamura et al.
molecular-weight form and that some substance
existing in the homogenate must have been essential for its conversion into the higher-molecularweight form. The existence of a renin-binding substance is therefore proposed and the mechanism of
its binding reaction with renin has been investigated.
In the present study, the molecular weight of
renin in the granules obtained by a newly devised
sieving method was measured, and an attempt
made to define the possible mechanism for the
molecular conversion of renin and the physicochemical properties of the renin-binding substance.
Materials and methods
Preparation of homogenate
Kidneys from mongrel dogs were excised under
sodium pentobarbital anaesthesia and immediately
washed with 100 ml of ice-cold saline solution by
gentle infusion into the renal artery. The renal
cortex was cut finely with a blade and transferred
to ice-cold sucrose (0.3 mol/l) (1 :9, w/v), after
which it was homogenized in a Potter-Elvehjem
homogenizer with a loose-fitting pestle at 800
rev./min for 45 s. After centrifugation at 500 g for
10 min, 10 ml of the homogenate was centrifuged
at 5000 g for 10 min to obtain a precipitate which
was subsequently resuspended in 10 ml of sucrose
(0.3 mol/l) and subjected to sieve separation. An
aliquot of the homogenate was centrifuged
separately at 100000 g for 30 min, the supernatant serving as ‘cytosol’ (Fig. I).
Sieving by membranejker
The apparatus used for isolating the renin
granules was a cylindrical sieve chamber, 3.5 cm
Kidney cortex
1
1
5009 for
Hornogentze I” sucrose I0 3 rnolill
Centrifuge a1
+
f
+
+
lorn1
lOml
Centrifuge at 50009 lor 10 min
Preclpllale
k----3
10 min
supernatan1
4
4
Resuspend with 10 ml 01 EUC~OS B10
diameter x 0.4 cm deep, made of Stirol. Both sides
of the chamber were fitted with a Nucleo-filter (0.6
,urn in pore size; Lot no. N-93-08, Nucleopore Corporation, California, U.S.A.). The chamber was
filled with 3.8 ml of the resuspended precipitate,
after which the whole apparatus was immersed in
ice-cold sucrose (0.3 mol/l). The same sucrose
solution was infused (0.15 ml/rnin) at a pressure of
30 mmHg into the chamber through an inlet tube
from a reservoir to wash out soluble materials and
intracellular particles smaller than renin granules.
The sieve chamber contained a magnetic stirrer.
One hour later, the contents of the chamber were
removed and centrifuged at 100 000 g for 30 rnin
to separate the supernatant and the sediment. The
activities of renin and other marker enzymes in
both fractions were determined and compared with
those in the supernatant and the precipitate
( 100 000 g x 30 min) from 3.8 ml of original
homogenate (3.8 ml of the original homogenate
was equal to the volume applied to the sieve
chamber). The renin activity of the supernatant of
the contents of the chamber was reduced to
approximately 3% of that of the supernatant of the
3 . 8 ml of homogenate, indicating that most of the
soluble renin in the homogenate could be washed
out by this technique. Similarly, the activities of
succinate dehydrogenase (a marker enzyme for
mitochondria) and acid phosphatase (lysosomes)
were almost 0’36, and the activity of glucose 6phosphatase (microsomes) was approximately
11%. The precipitate, however, showed some
activity of these enzymes. The renin activity of the
precipitate of the contents of the chamber was
approximately 64% of that of the precipitate from
3.8 ml of homogenate: this result means that 64%
of the renin granules could be separated from the
original homogenste. Similarly, approximately
36% of mitochondria, 3 1% of lysosomes and 8% of
microsomes were shown to exist as a contamination by measuring their respective marker
enzymes. The remaining contents of the sieve
chamber served as ‘renin granules’. The granules
were frozen and thawed five times and then
centrifuged at 100000 g for 30 min, the supernatant being used.
prec#w
Centrifuge a t 100 OOOg for
30 miri
3 mollli
Estimation of the molecular weights of renin and
(he renin-binding substance
3 em1
1
Piil in10 stwe chamber
FIG. I . Flow diagram for the preparation of renin
granules and cytosol.
The molecular weights of renin and the reninbinding substance were estimated by gel filtration
with Sephadex G-100 column and G-75 column
respectively ( I .6 cm diameter and 90 cm long). A
Characteristics of a renin-binding substance
phosphate buffer (0.05 mol/l) containing NaCl
(0.1 mol/l) (pH 7-2) was used for elution and 1 ml
fractions were collected at 4OC. The void volume
of the column was estimated by Blue Dextran, and
bovine serum albumin (molecular weight, 67 OOO),
ovalbumin (molecular weight, 45 OW), ctchymotrypsinogen A (molecular weight, 25 000)
and cytochrome c (molecular weight, 12 900) were
used as standards for molecular weights.
Enzyme activities
Tubes containing 0.1 ml portions of the sample,
0.1 ml of partially purified dog angiotensinogen
prepared by the method of Morimoto, Yamamoto,
Horiuchi, Tanaka & Ueda (1970) (the maximum
amount of angiotensin I that could be liberated;
200 pmol) and 0.8 ml of phosphate buffer (0.1
mol/l, pH 7.0), together with EDTA (10 mmol/l),
dimercaprol (3.2 mmol/l) and 8-hydroxyquinoline
sulphate (1.6 mmol/l), were incubated at 37OC for
30 min, and the reaction was stopped by immediate
cooling. The angiotensin 1 generated was determined by radioimmunoassay (CEA-IRE-SOLIN,
Italy) according to the method of Haber, Koerner,
Page, K l i a n & Purnode (1969).
Succinate dehydrogenase (EC 1.3.99.1) (for
mitochondria) was assayed by the method of Slater
& Bonner (1952); acid phosphatase (EC 3.1.3.2)
(for lysosomes) and glucose 6-phosphatase (EC
3.1.3.9) (for microsomes) were measured by the
method of Morimoto, Yamamoto & Ueda (1972),
in which p-glycerophosphate and glucose 6-phosphate were used as substrates respectively, and the
released inorganic phosphate was determined by a
0
modification of the method of Fiske & Subbarow
(1925) by Morimoto et al. (1970).
A cidiJication
Aliquots of cytosol from the renal cortex, and of
renin in the renin granules, were dialysed against
glycine/HCl buffer (0.05 mol/l) containing NaCl
(0.1 mol/l) (pH 3.0) for 24 h at 4°C and subsequently dialysed against phosphate buffer (0.05
mol/l) containing NaCl (0.1 mol/l) (PH 7.0) for 24
h at 4°C. As a control, the samples were dialysed
separately against phosphate buffer (0.05 mol/l)
(pH 7-0)for 48 h.
Inhibition of thiol groups
It has been suggested that thiol groups may be
involved in the molecular conversion of renin.
Inagami et al. (1977) and Funakawa et al. (1978)
reported that among several protease inhibitors,
sodium tetrathionate, which inactivates thiol
groups, was able to form the higher-molecularweight form of renin from kidney homogenate. We
used sodium tetrathionate as an inhibitor of thiol
groups to examine their possible role in the
conversion mechanism.
Results
Molecular weight of renin in the granules
Fig. 2 shows the gel-filtration profile of renin in
the granules. Inagami et al. (1977) reported that
sodium tetrathionate was essential for renin to
L
Elution volume (ml)
FIG.2. Elution profiles of renin in the granules with ( 0 ) and without ( 0 ) sodium tetrathionate
( 5 mmolh). A portion (1 ml) of the sample was applied to a Sephadex G-100 column (1.6 cm x 90 cm)
equilibrated with phosphate buffer (0.05 mol/l) containing NaCl (0.1 molh) at pH 7.2 and 1 ml
fractions of eluate were collected. Renin activity was expressed as pmol of egiotensin I (ANG I)
h-l xn-l.
347
348
M. Kawamura et al.
Elution volume (ml)
FIG.3. Gel filtration of the mixture of renin in the granules (0.5 ml) and cytosol (0.5 ml) with ( 0 )and
without ( 0 ) sodium tetrathionate (5 mmol/l). The mixture was applied to a Sephadex G-100 column
(1.6 cm x 90 cm) and eluted with phosphate buffer (0.5 mol/l) (pH 7.2) containing NaCl (0.I mol/l).
Fractions ( 1 ml) of eluate were collected. Renin activity was expressed as pmol of angiotensin 1 (ANG I)
h-' ml-'.
maintain its higher-molecular-weight form in the
kidney homogenate. However, the renin activity in
the granules appeared in the molecular-weight
region of 40 000 (lower-molecular-weight form of
renin), whether or not sodium tetrathionate (5
mmol/l) was added to the sample.
To examine the effect of cytosol on the
molecular conversion of renin, the cytosol (0.5 ml)
was mixed with renin in the granules (0.5 ml) 30
min before application to a Sephadex column at
4°C. When sodium tetrathionate (5 mmol/l) was
added to the mixture, the renin activity appeared in
the molecular-weight region of 60 000 (highermolecular-weight form of renin), and when sodium
tetrathionate was not added to the mixture, it
appeared in the region of 40000 (Fig. 3). These
results correspond to those of our previous experiments (Funakawa et al., 1978), in which the
granules were collected by discontinuous sucrosedensity-gradient centrifugation, clearly indicating
that renin stored in the granules is in the smaller
form, which is converted into the higher-molecularweight form by reacting with some substance in
cytosol of the renal cortex.
Esfect of p H and heating on the conversion of the
molecular weight of renin
The cytosol and renin in the granules were
separately dialysed against acid buffer for 24 h,
and then dialysed against neutral buffer for a
further 24 h. As a control they were dialysed
against neutral buffer for 48 h.
The mixture of either the acidified or the control
renin preparations with control cytosol (1 : 1, v/v)
in the presence of sodium tetrathionate (5 mmol/l)
resulted in the formation of the higher-molecular-
weight form of renin. On the contrary, the mixture
of either the acidified or the control renin preparations with acidified cytosol in the presence of
sodium tetrathionate (5 mmol/l) did not result in
the formation of the higher-molecular-weight form
of renin.
After the cytosol had been boiled for 10 min and
then mixed with the control renin preparation, the
molecular size of the renin was smaller despite the
presence of sodium tetrathionate. These results
suggest that a substance essential for the conversion of the lower- to the higher-molecular-weight
form of renin exists in cytosol, which loses its
activity if exposed to low pH or heat.
Separation of the renin-binding substance by
Sephadex column chromatography
A portion ( 1 ml) of the cytosol containing the
lower-molecular-weight form of renin was applied
on a Sephadex G-75 column to separate the eluate
into three fractions: fraction A from 57 (void
volume) to 75 ml, fraction B from 76 to 86 ml, and
fraction C from 87 t o 106 ml. The corresponding
molecular weight of the fractions was estimated as
shown in Fig. 4. Thus fraction A includes
substances of molecular weight over 47 000,
fraction B from 47 000 to 32 OOO, and fraction C
from 32 000 to 15 000. Each fraction was concentrated to 2 ml through a Diafilter membrane G1OT. Portions (0.3 ml) from the fractions were
mixed together and sufficient sodium tetrathionate
was added so that the final mixture had a tetrathionate concentration of 5 mmol/l. After the
mixture had been standing for 30 min at O°C, it
was applied to a Sephadex G-100 column to
estimate the molecular weight of renin. The gel-
Characteristics of a renin-binding substance
349
necessary for the molecular conversion from the
lower- into the higher-molecular-weight-form of
renin.
Discussion
c Fraction A
c
.FractionC
\
Hiqher-molecular-weight form of renin
Bovine serum albumin
2
3
4
10-4 x MOI. wt.
5
6
7
8
FIG.4. Estimation of molecular weight with Sephadex
G-75: fraction A (molecular weight: over 47000);
fraction B (molecular weight: 32 000-47 OOO); fraction
C (molecular weight: 15 000-32 000).
filtration profile showed a single peak of the highermolecular-weight form of renin, proving that a
substance essential for the molecular conversion of
renin was retained after gel filtration and subsequent concentration. Similarly, the mixing of 0.5
ml of fraction A and 0.5 ml of fraction B in the
presence of sodium tetrathionate resulted in the
higher-molecular-weight form of renin, whereas the
mixing of fractions B and C in the presence of
sodium tetrathionate resulted in the lowermolecular-weight form. These results indicate that
fraction C is not involved in the conversion
mechanism.
Since fraction B is rich in the lower-molecularweight form of renin, we next examined whether
renin in the granules could be substituted for
fraction B. A portion (0.5 ml) of the solution of
renin in the granules was mixed with 0-5 ml of
fraction A or of fraction B in the presence of
sodium tetrathionate and examined for molecular
conversion. The mixture of fraction A and renin
resulted in the higher-molecular-weight form; the
mixture of fraction B and renin resulted in the
lower-molecular-weight form of renin. It is thus
concluded that the substance essential for the
molecular conversion of renin is contained in
fraction A. Furthermore, the mixture of fraction A
and renin resulted in the lower-molecular-weight
form of renin when sodium tetrathionate was not
added, indicating that this potent thiol inhibitor was
Although numerous studies on the highermolecular-weight form of renin have been reported
for whole renal homogenate, little is known
regarding the renin in granules. The present study
indicates that stored renin in granules is in the lowmolecular-weight form. This molecular size did not
change even in the presence of sodium tetrathionate, but it changed to a higher-molecularweight form if the renin was mixed with cytosol in
the presence of sodium tetrathionate. This evidence
was similar to that obtained in studies in which the
renin granules were collected by sucrosedensitygradient centrifugation (Funakawa et al., 1978).
On the other hand, renal cortical cytosol is
thought to contain a renin-binding substance which
reacts with the lower-molecular-weight form of
renin to form a higher-molecular-weight form. An
acidification procedure allows the highermolecular-weight form of renin to be converted into
the lower-molecular-weight form. We previously
reported that acidified renal homogenate containing the lower-molecular-weight form of renin
failed to be converted into the higher-molecularweight form even if sodium tetrathionate was
present (Funakawa et al., 1978). In these experiments, we demonstrated that the renin-binding substance in cytosol was unstable at low pH, which
would possibly account for the irreversibility of the
acid conversion of renin. The activity of the reninbinding substance is also destroyed by heating.
These characteristics of the renin-binding
substance closely resemble those reported by Boyd
(1974) for pig kidney, and by Leckie & McConnell
(1975) for rabbit kidney. A conflicting result has
been presented with regard to the possible
molecular size of the renin-binding substance,
which was found to be 13 000 by Leckie &
McConnell (1975). In the present study the reninbinding substance was contained in fraction A
(molecular weight > 47 000) of the cytosol. However, such evidence would not necessarily indicate
the true molecular size of the substance. Since the
difference in molecular weight between the higherand the lower-molecular-weight forms of renin is
20000, it is possible that the renin-binding substance usually exists as an aggregated form, one
cluster having a molecular weight of 20 000.
Alternatively, the molecular size may have been
350
M . Kawamura et al.
overestimated by gel filtration, which method is frequently subject to error in the estimation of the
molecular weight of glycoproteins or non-spherical
substances.
The mode of the conversion reaction is not
obvious. Is endogenous protease involved in the
conversion mechanism? Various reports have suggested that inactive renin is activated by trypsin
(Morris & Lumbers, 1972; Cooper, Murray &
Osmond, 1977) or kallikrein (Sealey, Atlas,
Laragh, Oza & Ryan, 1978). Soya-bean trypsininhibitor (Derkx, Tan-Tjong & Schalekamp, 1978)
and trasylol (Aprotinin, Bayer) (Derkx et al., 1978;
Leckie, 1978), an inhibitor of kallikrein, have been
cited as inhibitors of the activation of inactive
renin. If protease is involved in the molecular
conversion of renin, the enzyme would be contained in fraction A as well as the renin-binding
substance. Sodium tetrathionate may act as a
protease inhibitor. On the other hand, the interconversion may not involve an enzymic reaction. In
this case, if the renin-binding substance and renin
have active thiol groups, sodium tetrathionate may
act as a thiol oxidant and bind renin and the reninbinding substance with disulphide bonds. Thiol
oxidation thus appears necessary for the formation
of the higher-molecular-weight form of renin,
whichever of the two possibilities for the conversion
system mentioned above obtain. An appropriate
explanation may arise from further studies with a
more purely extracted renin-binding substance and
renin.
Acknowledgment
We thank Miss Masako Yamada for providing
skilful technical assistance.
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