Pseudorenin
A NEW ANGIOTENSIN-FORMING ENZYME
By Leonard T. Skeggs, Ph.D., Kenneth E. Lentz, Ph.D.,
Joseph R. Kahn, M.D., Frederic E. Dorer, Ph.D.,
and Melvin Lerine, Ph.D.
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ABSTRACT
A new enzyme, pseudorenin, has been discovered which resembles renin in
its ability to form angiotensin I from the synthetic tetradecapeptide renin
substrate and from purified hog renin substrate A. Its maximal activity occurs
at a much lower pH value than does that of renin. The two enzymes may be
easily separated by DEAE-cellulose chromatography. Unlike renin, pseudorenin does not attack substrate A in the presence of serum, nor does it produce
angiotensin I from renin substrate as it exists in serum. In contrast to renin,
which occurs primarily in the kidney, pseudorenin has been found in every one
of the 13 different tissues which have been tested, and also in the plasma. The
natural substrate for the new enzyme as well as its physiological function are
not known.
ADDITIONAL KEY WORDS
• The enzyme renin is the primary activator
of the renin-angiotensin pressor system. It is
found in the kidney, from which it may be
secreted into the bloodstream, where it
hydrolyzes a protein substrate releasing the
decapeptide angiotensin I. A converting enzyme cleaves this latter peptide, removing the
dipeptide His-Leu from its C-terminal, thus
producing angiotensin II, a powerful vasoconstrictive compound which is the effector
substance of the system (1).
The protein substrate for renin was purified
from hog plasma (2). Five major forms were
found: A, B1; B2, Ci, and C2. All were
glycoproteins with molecular weights of about
57,000. The renin substrate from horse plasma
was degraded with trypsin to yield a peptide
fragment which yielded angiotensin I on
further treatment with renin. The fragment, a
tetradecapeptide, was isolated and its structure determined and confirmed by synthesis
(3, 4). The kinetics of the reaction of hog
From the Department of Medicine and Surgery,
Veterans Administration Hospital, and the Departments of Biochemistry and Pathology, Case Western
Reserve University, Cleveland, Ohio 44106.
Received July 15, 1969. Accepted for publication
August 26, 1969.
Circulation Research, Vol. XXV,
October 1969
pseudorenin
renin
renin with this tetradecapeptide and with a
number of related synthetic peptides were
recently described (5).
A new enzyme has now been found which
produces angiotensin I from the tetradecapeptide renin substrate and from purified hog
renin substrate A. It differs from renin in that
it does not produce angiotensin I from the
substrate occurring naturally in plasma, nor
does it attack the purified substrate A in the
presence of plasma. It is chromatographically
distinct from renin, and has optimum enzymatic activity at much lower pH values. Its
activity has been found in every tissue thus far
tested. This paper contains a description of
the new enzyme, which has been named
pseudorenin.
Experimental
Methods of Assay.—Samples to be assayed
for pseudorenin were diluted to 1 ml with icecold saline in siliconized Pyrex tubes. One ml
of a cold solution containing 1 nmole of
tetradecapeptide substrate was then added.
The substrate solution was prepared in 0.05M
sodium citrate buffer, pH 4.0, containing 0.1M
NaCl. The tubes were then incubated at 37.5°
for 15 minutes. The incubation, as well as all
451
452
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others described in this paper, was terminated
by the addition of 1 ml (in some cases 2 ml)
of a solution containing NaH2PO4 and HC1 or
Na2HPO4 and NaCl to adjust the pH to 5.5
and yield approximate isotonicity. The tubes
were then heated on a boiling water bath for
10 minutes, cooled, and centrifuged if necessary. The supernatant fluid was assayed in the
rat using a standard solution of angiotensin I
(6).
The tetradecapeptide itself gives a small,
but significant, pressor response in the test
animal. In order to correct for this effect,
control tubes, complete except for enzyme,
were prepared for each set of assays. The
substrate preparation was purposely kept low
to minimize this blank and also to conserve
the synthetic substrate. The amount of angiotensin generated was nearly proportional to
the enzyme concentration when less than 30%
of the substrate was consumed.
Human renin assays were conducted in an
analogous fashion. Samples were diluted to 1
ml in cold saline in siliconized tubes. One ml
containing 1 nmole of human protein substrate was added. The substrate was dissolved
in 0.05M sodium phosphate buffer containing
0.1M NaCl and having a pH of 7.5. The tubes
were mixed and incubated at 37.5° for 15, 30,
or 60 minutes, depending on the sensitivity
required. The incubation was terminated and
assays performed as indicated above.
In previous work from this laboratory,
similar assays were performed on aliquots of a
standard renin preparation which allowed
calculation of results in terms of Goldblatt
units (6). It was necessary in this work to
compare the renin and pseudorenin activities
of various preparations. Since a standard
pseudorenin preparation was not available, it
was more meaningful to determine activities
of both enzymes in terms of nmoles of
angiotensin I produced per hour per milliliter
of enzyme preparation. One unit of renin or
pseudorenin activity was defined as that
amount of enzyme which produced 1 /xmole
of angiotensin I in 1 hour under the conditions
described above.
The pseudorenin assays were performed at
SKEGGS, LENTZ, KAHN, DORER, LEVINE
pH 4.0 rather than at the optimum pH 4.5 to
reduce to a minimum the activity of any renin
which might be present in the preparations.
The human renin assays were conducted at
pH 7.5 rather than at the optimum pH 6.0 to
reduce as far as possible the activity of the
pseudorenin contained in the preparation.
Preparation of Protein Renin Substrate —
Human citrated plasma (outdated bank
blood) was adjusted to pH 6.0 and diluted to
4% protein.1 An equal volume of cold 3M
ammonium sulfate was added slowly with
stirring. The precipitate was removed. A
sufficient amount of dry, solid ammonium
sulfate was added to raise the concentration of
the salt to 2.5M. The resulting precipitate was
gathered by centrifugation, dissolved in water,
and dialyzed first against 0.003M EDTA and
1% NaCl at pH 7.0, and then against distilled
water. The thoroughly dialyzed material was
centrifuged, and the supernatant fluid lyophilized. The final product, when dissolved in the
appropriate buffer, was angiotensinase-free,
and contained 70 nmoles of renin substrate
activity per gram of protein.
Preparation of Tetradecapeptide Substrate.
—The peptide was synthesized by the solid
phase method as described by Marshall and
Merrifield for the synthesis of angiotensin II
(7). Resin, 12.2 g, substituted with 5 mmoles
of tBoc-O-Bz-Ser was used as the starting
material. Although radioactive labeling was
not needed in the present work, 0.717 mmoles
of randomly labeled »C tBoc-Leu (3.06 X 10°
dpm) was introduced in the third step. After
adequate reaction time had been allowed, the
usual 20 mmoles of unlabeled tBoc-Leu was
introduced. It was found by difference that
72% of the radioactive amino acid was
incorporated. After all 13 cycles of the process
were completed, the crude peptide (5.38 g or
2.71 mmoles) was removed from the resin by
HBr in trifluoroacetic acid. The material was
dissolved in a mixture of 180 ml of methanol
and 20 ml of 1.7M acetic acid. Fifty-four g of
palladium was added and the mixture treated
1
Proteins were determined by an automated
modification of the Lowry method as previously
described (2).
Circulation Research, Vol. XXV, October 1969
NEW ANGIOTENSIN-FORMING ENZYME
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with hydrogen at 50 psi for 60 hours. At this
time, reduction was complete and a total of
0.295 mmoles of substrate activity (11.8$ of
the crude peptide) was found by biological
assay.
A portion of the material (0.15 mmoles)
was purified by two countercurrent distributions—first in a system formed of 0.01M
NaoCO.rNaHCO3 buffer, pH 10.5, and secbutyl alcohol, and second in one formed of
O.OIN HCl-2% NaCl and sec-butyl alcohol. The
distribution coefficient of the peptide in the
latter system was 2.69 after 600 transfers,
which corresponds closely to the value of 2.78
obtained for the compound first synthesized in
this laboratory (4). We obtained 0.072
mmoles of purified product (5.09 X 108 dpm
per mmole). This gave the expected amino
acid analysis, and was indistinguishable from
a sample of the original product when
chromatographed simultaneously on paper
(Rf 0.60 in n-butyl alcohol-acetic acid-water
[4, 1, 5] Whatman no. 1 paper), or by
biological assay after treatment with an excess
of renin. The original synthetic product began
to decompose at 219° to 220°, and liquefied at
249° to 250°; the present material melted at
255° to 258° with decomposition.
Preparation of Renin and Pseudorenin.—
After removal of all fat and connective tissue
4.8 kg of human kidneys was passed through
an electric meat grinder into 4.8 liters of water
at room temperature. The mixture was stirred
for 1 hour and then strained through gauze.
The kidney pulp was extracted with a second
4.8-liters of water for another hour and the
mixture was strained once more. The pulp was
discarded and the two filtrates were combined, chilled to 5°, and centrifuged2 for 2
hours (all procedures from this point forward
were carried out at refrigerator temperatures ).
The supernatant fatty pad, and the gray
precipitate were discarded. The turbid, red
extract with a volume of 8.28 liters contained
a total of 180 g of protein. It was diluted with
2
Intemational serum centrifuge Model No. 13L.
International Equipment Co., Boston, Mass.
Circulation Research, Vol. XXV,
October 1969
453
49.2 liters of cold, distilled water, and 2,710 g
of a moist filter cake of the free base form of
DEAE-cellulose (diethylaminoethyl cellulose)
was then added and the mixture stirred for 30
minutes. During this period, the pH was held
at 8.7 by the addition of small amounts of 2.5N
HC1. The DEAE-cellulose was removed by
filtration through gauze on a large vacuum
funnel. The adsorbent was thoroughly
squeezed by the use of a rubber dam covering
the funnel (filtrate = fraction A). The
DEAE-cellulose pad was stirred 10 minutes
with 50 liters of cold water and refiltered
(fitrate = fraction B). The DEAE-cellulose
pad was suspended in 16.6 liters of cold water
and the pH adjusted to 4.8 with 450 ml of IN
acetic acid. After 10 minutes of stirring,
the mixture was filtered (filtrate = fraction
Ci). The pad was washed by stirring for
10 minutes with 16.6 liters of cold water and
filtered (filtrate = fraction C 2 ). The pad was
stirred once more with 16.6 liters of cold
water. Solid NaCI, 97 g, was added, followed
by 2 liters of IN acetic acid, bringing the
mixture to pH 4.0. After 10 minutes of stirring,
the mixture was filtered (filtrate = fraction
D a ). The pad was suspended once more in
16.6 liters of water, and 97 g of NaCl and
300 ml of IN acetic acid were added to bring
the mixture to pH 4.0. After 10 minutes of
stirring, the final filtration was accomplished
(filtrate = fraction D 2 ) and the pad set aside.
Renin and pseudorenin assays as well as protein determinations were performed on all of
the filtrates (Table 1).
Fractions Ci and C2 were combined, as
were Y)x and D2. Sufficient dry, solid
(NH4)2SO4 was dissolved in each to raise the
concentration of the salt to 2.5M. The resulting
precipitates were collected by centrifugation,
dissolved in water, and dialyzed against cold,
distilled water (fractions C and D).
As shown in Table 1, the foregoing
procedure yielded one fraction containing a
preponderance of pseudorenin (fraction C)
and another containing the major portion of
the renin (fraction D). Both fractions were
relatively free of angiotensinase, which might
interfere with assays. They were further
SKEGGS, LENTZ, KAHN, DORER, LEVINE
454
TABLE 1
Renin and Pseudorenin Fractions
Fraction
A
B
c,
Di
D.,
c"
D
E
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F
G
Description
Filtrate from original
adsorption
Neutral wash
pH 4.8 eluate
pH 4.8 wash
pH 4.0 eluate
pH 4.0 wash
pH 4.8 eluates, combined,
concentrated and dialyzed
pH 4.0 eluates, combined,
concentrated and dialyzed
Pseudorenin peak from chromatography of
fraction C concentrated
Final pseudorenin preparation
Final renin preparation
purified by DEAE-cellulose chromatography.
The chromatography column, 2.6 cm i.d.
and 100 cm long, was packed with DEAEcellulose (Whatman DE 32) which had been
equilibrated with 0.005M sodium phosphate
buffer at pH 7.5. Fraction C, containing the
pseudorenin, was adjusted to pH 7.5, a small
precipitate removed, and the clear solution
with a volume of 590 ml was pumped onto the
column at the rate of 1.0 ml/min. After the
sample was applied, the 0.005M phosphate
(pH 7.5) buffer was pumped from a 500-ml
gradient mixing chamber onto the column at
the same rate of 1 ml/min. At the same time,
0.025M sodium acetate buffer having a pH of
5.4 was pumped into the chamber at 1
ml/min, thus forming a descending pH
gradient. Ninety-five fractions of 25 ml were
collected and assayed for renin, pseudorenin,
and protein. The renin assays were negative
throughout. The principal portion of the
protein did not adsorb on the column, but
simply passed through. The pseudorenin
activity was found in a single symmetrical
peak, which was eluted during a period of
increasing conductivity, but at the original pH
and lying between tubes 51 and 72. These
fractions were pooled (fraction E) and
lyophilized. The dried material was dissolved
in water, adjusted to pH 5.0, and clarified by
centrifugation, yielding 32 ml of the final
Pseudorenin
(units)
Renin
(units)
Protein
2180
2.2
4480
1540
1170
250
1.11
0.60
1.20
0.60
20.00
4.00
63.2
1.65
40.7
6.0
34.2
7.1
5500
1.89
4.0
1140
10.70
18.3
1080
607
11.9
0
0
2.37
(g)
0.334
0.236
7.5
pseudorenin preparation (fraction F), which
assayed 19 units/ml and contained a total of
607 units, with a specific activity of 2.6
units/mg of protein.
The pH 4.0 eluate (fraction D), which
contained the principal portion of the renin,
was chromatographed on the same DEAEcellulose column used for pseudorenin. The
adsorbent in this case was equilibrated with
0.025M sodium acetate buffer, pH 5.4. The
sample was adjusted to this pH and conductivity, and the precipitate which formed was
removed by centrifugation and washed with
column buffer. The clarified sample and wash
were combined. The preparation measured
965 ml and contained 17.6 g of protein. It was
applied to the column at 0.8 ml/min. After
application of the sample, the column was
developed with 1 liter of 0.025M sodium
acetate buffer at pH 5.4. This was followed by
0.025M sodium acetate, pH 4.0, containing
0.1M NaCl. The major portion of the pseudorenin together with 40% of the renin and a
large amount of protein washed through
without adsorbing. Fractions 93 to 105, which
occurred during a steeply rising conductivity
gradient, contained a single sharp renin peak
together with a large amount of protein but a
relatively small amount of pseudorenin. The
fractions were pooled (fraction G) to form
the final renin preparation, which was kept in
Circulation Research, Vol. XXV,
October 1969
NEW ANGIOTENSIN-FORMING ENZYME
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the frozen state until used; 2.37 units were
obtained, with a specific activity of 0.0032
units/mg of protein.
The recovery of both pseudorenin and renin
from these procedures was very disappointing.
However, it was of primary importance in this
work to separate the two activities. Thus
pseudorenin was obtained completely free of
renin, although in only a 20% yield. A further
loss was incurred during the simple lyophilization of the preparation.
The renin purification was also attended by
extremely high loss. Yet the final preparation
obtained has as low a ratio of pseudorenin to
renin (5.0) activity as it has thus far been
possible to obtain. Since the activity of
pseudorenin is approximately 1360 times
greater at pH 4.0 than at pH 7.5, the
preparation is essentially free of this activity
at the latter pH.
Structural Requirements of a Substrate for
Pseudorenin.—One-fA aliquots of the pseudorenin preparation (approximately 0.015 units)
were incubated for 16 hours with 0.5-ml
samples containing 500 nmoles of each of
several different synthetic peptides (5). The
incubation mixtures were maintained at pH
4.0 by adding 0.05 ml of 0.1M sodium citrate
buffer. At the end of the incubation period,
the reactions were stopped by heating on a
boiling water bath for 10 minutes. Fifty-jid
samples of each of the incubation mixtures
representing 50 nmoles of the original peptide
were chromatographed on Whatman no. 1
paper, using n-butyl alcohol-acetic acid-water
[4, 1, 5].
No spots of any kind were found on
chromatography of a control incubation mixture containing the enzyme and buffer, but
without peptide.
The incubation mixture, using the tetradecapeptide substrate, yielded a ninhydrin-positive, Durrum-positive (His) spot at Rf 0.45,
corresponding to angiotensin I as well as a
ninhydrin-positive, Durrum-negative spot at
Rf 0.74, corresponding to Leu-Val-Tyr-Ser.
There was only a very faint Durrum-positive
spot at Rf 0.55, corresponding to the uncleaved tetradecapeptide, and the hydrolysis
Circulation Research, Vol. XXV,
October 1969
455
of the peptide was therefore virtually complete. A sample of the peptide His-Pro-PheHis-Leu-Leu-Val yielded a single yellow
ninhydrin-positive, Durrum-positive spot at
Rf 0.69, which exactly corresponded to control
spots of the unincubated peptide. A sample of
the peptide Leu-Leu-Val-Tyr#OEt yielded a
single pink ninhydrin-positive spot at Rt 0.92,
which also corresponded to control spots of
the unincubated peptide.
The ability of the enzyme to cleave the
tetradecapeptide, the nonapeptide His-ProPhe-His-Leu-Leu-Val-Tyr-Ser, and its inability
to cleave His-Pro-Phe-His-Leu-Leu-Val and
Leu-Leu-Val-Tyr'OEt were confirmed by direct determination by the ninhydrin method
(5) of new amino groups formed during
incubation.
Solutions containing 200 nmoles each of the
peptides in 10-ml portions of 0.01M sodium
citrate buffer at pH 4.5 were chilled in ice, 10jjl aliquots of pseudorenin (0.19 units) were
added, and the solutions mixed. The amino
groups initially present in these mixtures were
determined without delay by direct sampling
into the AutoAnalyzer. The tubes containing
the mixtures were then incubated at 37.5°, and
were sampled several times during the following 100 minutes.
,
The enzyme produced 132 nmoles of new
amino groups from the tetradecapeptide; the
initial rate being in excess of 30 nmoles/ml/
hour. From His-Pro-Phe-His-Leu-Leu-Val-Tyr
Ser 86 nmoles were produced at an initial rate
of 13.2 nmoles/ml/hour. In contrast, a questionably significant 11.0 nmoles were formed
from His-Pro-Phe-His-Leu-Leu-Val at the very
slow rate of 0.66 nmoles/ml/hour. No new
amino groups were produced from Leu-LeuVal-Tyr'OEt.
The experiments show that pseudorenin will
hydrolyze the nonapeptide His-Pro-Phe-HisLeu-Leu-Val-Tyr-Ser, but will not attack the
peptide His-Pro-Phe-His-Leu-Leu-Val. This is
in accordance with the structural requirements
for a renin substrate (5). Taken together with
the failure of the enzyme to hydrolyze LeuLeu-Val-Tyr*OEt and its inability to degrade
angiotensin I, it would indicate that pseudo-
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renin is not a simple leucylleucinase, but is a
highly specific endopeptidase with structural
requirements for its substrate that are remarkably similar to renin.
Products of the Action of Pseudorenin on
the Tetradecapeptide Substrate.—A 5-//.1 aliquot of a pseudorenin preparation (0.107
units) was added to 0.5 ml of a solution
containing 500 nmoles of tetradecapeptide.
The pH was adjusted to 4.5 and the
unbuffered mixture incubated for 7 hours at
38°. One-yul samples were withdrawn at
hourly intervals, diluted in saline, heated on
the boiling water bath for 10 minutes and
assayed in the rat. Pressor activity equivalent
to 400 nmoles of angiotensin I was found at
the end of 1 hour. No further change was
detected during the ensuing 6 hours of
incubation. At the end of the incubation, 0.4
ml of the mixture was applied to Whatman
no. 1 paper as a 2-cm streak and chromatographed with n-butyl alcohol-acetic acidwater [4, 1, 5]. A 3-mm strip cut from the
center of the developed chromatogram revealed a ninhydrin-positive, Durrum-negative
spot with an Rf of 0.74, corresponding to LeuVal-Tyr-Ser, and a single ninhydrin-positive,
Durrum positive spot Rf 0.48, corresponding
to angiotensin I. Relatively minor ninhydrinpositive spots with values of 0.17, 0.24, and
0.30 were identified from control chromatograms as impurities in the enzyme preparation.
The sections of the chromatogram corresponding to angiotensin I and to Leu-Val-TyrSer were eluted with O.IN acetic acid to permit
their identification. The eluate of the band at
Rf 0.48 contained pressor activity equal to 77
nmoles of angiotensin I (33% of theory). A
hydrolysate prepared by heating with 1.0 ml
of 6N HC1 in an evacuated, sealed tube for 22
hours revealed the following amino acid molar
ratios upon Dowex 50 chromatography: Asp
0.35, Arg 0.89, Val 0.86, Tyr 0.98, lieu 0.69,
His 1.31, Pro 0.97, Phe 0.46, and Leu 1.00.
Relatively minor peaks with molar ratios
ranging from 0.04 to 0.22 were observed for
Thr, Ser, Glu, Gly and Ala.
The pressor product was further identified
SKEGGS, LENTZ, KAHN, DORER, LEVINE
by its behavior in a 10-tube countercurrent
distribution in the system .sec-butyl alcohol0.1M sodium phosphate at pH 7.0 containing
15% NaCl. Of the pressor activity, 94% was
found in tubes 4 to 9, and only 6% in tubes 0 to
3, a distribution which corresponds to that of
pure angiotensin I.
The eluate of the band at Rf 0.74
corresponding to Leu-Val-Tyr-Ser contained
radioactivity equivalent to 151 nmoles (65% of
theory) of tetradecapeptide. Hydrolysis and
amino acid chromatography yielded the following molar ratios: Leu 1.00, Val 0.80, Tyr
0.67, and Ser 1.05. Minor peaks with molar
ratios between 0.10 to 0.38 were found for
Asp, Glu, Gly and Ala.
Although the molar ratios obtained in these
experiments are disappointingly far from
being whole numbers, the indicated composition of the two bands correspond to those of
angiotensin I and Leu-Val-Tyr-Ser. The countercurrent distribution confirms the identity of
the pressor product as angiotensin I rather
than angiotensin II. Finally, it should be
noted that the pressor product was formed
during the first hour of incubation and that no
loss of pressor activity and presumably no
further degradation occurred during the next
6 hours.
Relationship of Activity and pH for Renin
and Pseudorenin.—Aliquots of pseudorenin
solution, free of renin activity and ranging
from 2 fxl to 500 fj\ in volume (0.312 units/ml)
depending on the expected activity, were
diluted to 1.0 ml with cold saline. One ml of a
solution containing 1 nmole of the tetradecapeptide substrate in 0.025M sodium citrate,
0.075M sodium phosphate buffers with pH
values from 3.0 to 8.5 at 0.5-unit intervals
were added. The tubes were mixed and
incubated at 37.5° for 15 minutes. The
incubations were terminated and the solutions
assayed as described above. The results,
shown in Figure 1, reveal that maximal
activity occurs at pH 4.5, and that there is a
pronounced shoulder in the curve at pH 6.0.
The velocity drops to small but easily
measurable values at pH 7.0 and above.
Portions of several buffer solutions measurCirculation Research, Vol. XXV, October 1969
NEW ANGIOTENSIN-FORMING ENZYME
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pH
FIGURE 1
Effect of pH on the action of pseudorenin on the tetradecapeptide substrate.
ing 4.75 ml each, with pH values ranging from
3.5 to 8.0 at 0.5-unit intervals and composed of
0.0125M sodium citrate, 0.0375M sodium
phosphate, and 0.077M NaCl were placed in
tubes and chilled in an ice bath. Fifty /xl
containing 5.4 mnoles of purified hog substrate A were added, followed by 0.2 ml of
diluted solutions of pseudorenin (0.95 units).
The solutions were mixed and incubated at
37.5° The reaction was terminated in 1.0-ml
aliquots withdrawn at 0, 15, 30 and 60
minutes. The assay results showed that the
angiotensin was liberated at all pH values at
rates which were nearly linear over the 1-hour
incubation period. The initial rates are plotted
in Figure 2. The curve has a very sharp
maximum at pH 5.0 which drops to zero at pH
7.0 and above.
Aliquots of a human renin preparation
(0.0037 units/ml of renin and 0.032 units/ml
of pseudorenin) between 5 and 200 /A in
volume, depending on the expected activity,
were diluted to 1.0 ml in cold saline. One-ml
portions of a 0.025M sodium citrate-0.075M
phosphate buffer ranging in pH from 3.0 to
Circulation Research, Vol. XXV,
October 1969
457
8.5 and each containing 1 nmole of partially
purified human protein substrate were added.
The solutions were mixed and incubated for 3
hours at 37.5°. The reactions were terminated
as before and the solutions assayed. The
results are presented graphically in Figure 3,
and show that the maximal activity occurs at
pH 6.0. This is in substantial agreement with
other reports (8). There is a shoulder in the
curve at pH 6.5. The velocity at pH 7.5 is a
little more than half (54%) of its maximum
value at pH 6.0.
The renin preparations thus far obtained
have not been sufficiently free of pseudorenin
to determine the effect of pH on the
hydrolysis of tetradecapeptide substrate by
renin acting alone. The activity of renin on the
natural protein substrate can be observed,
since pseudorenin does not attack the crude
substrate prepared from serum.
Reaction Kinetics for Renin and Pseudorenin.— Two sets of 0.9-ml aliquots of the
semipurified human substrate preparation
ranging in concentration from 0.388 to 3.88
/AM were prepared in a buffer composed of
0.1M NaCl and 0.05M sodium phosphate, pH
7.5. One-tenth ml of a human, angiotensinase-
pH
FIGURE 2
Effect of pH on the action of pseudorenin on purified
hog substrate A.
SKEGGS, LENTZ, KAHN, DORER, LEVINE
458
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'/s
FIGURE 4
FIGURE 3
Effect of pH on the action of human renin on human
protein substrate.
free renin8 preparation suitably diluted in the
buffer and containing 0.02 Goldblatt units was
added to one set, and 0.04 Goldblatt units was
added to the other set. The mixtures were
incubated at 37.5° for 30 minutes. The
reactions were terminated and the solutions
assayed. The data are presented in Figure 4.
Similar dilutions of 0.9-ml portions of
tetradecapeptide substrate were prepared in
0.05M sodium citrate, 0.1M NaCl buffer with
pH 4.5. One-tenth ml of pseudorenin solution
diluted in buffer was added to each. The
dilutions of the enzyme yielded a final
pseudorenin concentration of 3.0 X 10"4 units/
ml of reaction mixture in one case, and
6.0 X 1(H units/ml in the second set. The
mixtures were incubated for 15 minutes, the
reactions terminated, and the assays performed as before. The data are presented in
Figure 5.
Similar efforts were made to determine the
kinetics of the reaction of pseudorenin with
purified hog substrate A in 0.05M citrate, 0.1M
3
The human renin used in this experiment was very
kindly supplied by Dr. Erwin Haas of the L. D.
Beaumont Memorial Laboratories, Mt. Sinai Hospital,
Cleveland, Ohio.
Lineweaver-Burk plots for the hydrolysis of human
protein substrate by human renin at pH 7.5. Renin
concentration in Goldblatt units per ml: solid circles
= 0.04; open circles = 0.02. Substrate concentration
in M X 10°, and velocity in M X 106/min. Average
Km = 1.32 x 10-" M; average Vm = 0.75 X 10-"M/
min/Goldhlatt unit.
FIGURE 5
Lineweaver-Burk plots for the hydrolysis of tetradecapeptide substrate by pseudorenin at pH 4.5. Substrate
concentration in M X 10e, and velocities in M X 10"/
hr. Pseudorenin concentration: solid circles = 3.0 X
10-'< units/ml; open circles = 6.0 X 10-* units/ml.
Average Km = 1.85 X 10-° M; average Vmax = 8.2x
lO-iiu/hr/unit of pseudorenin.
Circulation Research, Vol. XXV, October 1969
NEW ANGIOTENSIN-FORMING ENZYME
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FIGURE 6
Inhibition of the action of pseudorenin
bij serum. Solid circles = pseudorenin
open squares = pseudorenin + serum;
= pseudorenin -f- substrate A -f serum;
serum -f- tetradecapeptide substrate.
on substrate A
+ substrate A;
solid squares
open circles =
sodium chloride at its pH optimum of 5.0. In
five trials, Km values ranging from 0.66 to
2.49 X 10-°M were obtained. The affinity of
the enzyme for this highly purified protein
substrate is in the same order of magnitude as
its affinity for the tetradecapeptide substrate
(Km = 1.85 X 10" 6 M). Its affinity is also similar
to that of renin for its protein substrate at pH
7.5(Kin = 1.32xl0-°M).
The Vmax (maximum velocity) for the
reaction of pseudorenin with the tetradecapeptide substrate is 8.2 X 10~3M/hour for
each unit of enzyme while the corresponding
figure for its hydrolysis of substrate A was
approximately 3.6 X 10~6M/hour. The velocity
of the reaction with the tetradecapeptide is
therefore 2,280 times faster than with substrate A.
Effect of Serum on the Reaction of
Pseudorenin with Substrate A.—A nonhemolyzed hog serum was thoroughly dialyzed
against distilled water and the insoluble material removed by centrifugation. One-tenth
volume of a solution containing 0.5M sodium citrate and 1.0M sodium chloride was
added and the pH adjusted to 4.0. A 5-ml
Circulation Research. Vol. XXV,
October 1969
459
sample of the serum was incubated with 1
nmole of angiotensin I for 1 hour. There was
no loss of pressor activity, and the serum was
considered free of angiotensinases under these
conditions.
Fifty jul of purified substrate A (5.4
nmoles) was diluted with 4.85 ml of buffer,
and 0.1 ml (1.9 units) of pseudorenin was
added. The tubes containing the mixture were
incubated at 37.5° for 15 minutes. The
reaction was terminated in 1.0-ml samples at
0, 7.5 and 15 minutes. The results of the assays
are shown in Figure 6 (solid circles) and
demonstrate the ability of pseudorenin to
generate angiotensin from purified hog substrate A.
An exactly parallel experiment was performed in which 0.1 ml of pseudorenin was
incubated with 4.9 ml of the whole hog serum
prepared above. The results of the assays of
this mixture are shown by the open-square
curve (Fig. 6) and illustrate the inability of
the enzyme to produce angiotensin from the
substrate contained in hog serum.
The addition of whole serum (4.85 ml)
rather than buffer to the mixture of pseudorenin (0.1 ml) and substrate A (50 fjA)
reduced the production of angiotensin nearly
to zero as illustrated by the solid-square
curve.
A fourth incubation mixture was prepared
in which 5 jul (5.0 nmoles) of tetradecapeptide substrate was added to 5 ml of the serum.
The generation of angiotensin by this mixture
is illustrated by the open-circle curve, which
suggests that the endogenous pseudorenin in
the hog serum is capable of hydrolyzing the
tetradecapeptide substrate in the presence of
the serum inhibitor.
These experiments suggest very strongly
that hog serum inhibits the hydrolysis of
substrate A by pseudorenin. The fact that
serum does not prevent the production of
angiotensin from the tetradecapeptide substrate by the endogenous pseudorenin in
serum would suggest the possibility that the
inhibitory action is against the natural protein
substrate and not the enzyme.
Occurrence of Pseudorenin.—A male rat
SKEGGS, LENTZ, KAHN, DORER, LEVINE
460
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weighing 410 g was anesthetized with sodium
amytal, 9 mg/100 g ip. The right jugular vein
was catheterized and 0.3 ml (3.0 mg) of
heparin administered by this route. The left
carotid artery was cannulated and the animal
exsanguinated. The blood was chilled and
centrifuged and the clear plasma collected.
The organs were dissected free of connective
tissue, blotted, weighed, and chilled without
delay. The tissues were ground with 2 volumes
of water, using a Teflon-and-glass or all-glass
homogenizer. Four volumes of water were
used for the aorta and thymus, and 10
volumes for the adrenals because the weights
of these organs were small. The homogenates
were centrifuged and the residues discarded.
The supernatant fluids were kept in the frozen
state until needed.
Samples, which varied in size from 2 fi\ in
the case of the salivary gland to 10 fi\ in the
liver extract (1 ml of plasma was used), were
diluted to 5 ml in 0.05M sodium citrate, 0.1M
NaCl with pH 4.0 and 5.0 nmoles of the
tetradecapeptide substrate. The mixtures were
incubated at 37.5° for 30 minutes. One-mi
samples were removed at 0, 7.5, 15, and 30
minutes, the reaction was terminated, and the
supernatant fluid was assayed in the rat.
Angiotensin was produced in all cases at rates
which were usually nearly linear with time
and not suggestive of the presence of angiotensinase. Exactly similar assays in which the
incubation mixtures contained 0.01M EDTA
Spl..n
and 0.003M diisopropyl fluorophosphate were
also performed. In no case did the addition of
these angiotensinase inhibitors increase the
amount of angiotensin produced. The results
of this experiment are illustrated in Figure 7.
It is apparent that simple aqueous extracts
of all of the tissues tested possess the ability to
produce angiotensin from the tetradecapeptide substrate. It is all the more remarkable
that 2-fil aliquots of such extracts were
adequate to demonstrate this ability in many
of the tissues.
Presence of Pseudorenin in Human Plasma.
—The angiotensinase in a number of serum
samples was destroyed by chilling to 4° and
acidifying to pH 1.5 with HC1. After 10
minutes, the pH was adjusted to 7.0 and
samples were dialyzed for 16 hours against 1%
NaCl containing 0.003M EDTA.
Samples of serum, 0.5 ml, were mixed with
0.05 ml of 0.5M sodium citrate buffer with a
pH of 4.5, 1.8 ml of saline, 0.02 ml of 0.3M
diisopropyl fluorophosphate, and 0.13 ml of
1.0 HIM of tetradecapeptide subtrate. The
tubes containing the mixtures were incubated
at 37.5° for 15 minutes. The pH of the
solution was adjusted to 5.5, and 7.5 ml of
water was added. The tubes were heated on
the boiling water bath for 10 minutes and
centrifuged, and the supernatant fluid was
assayed in the rat.
Renin assays were performed on the same
serum samples by the method of Gould et
al. (9).
The results of the pseudorenin and renin
assays are shown in Table 2 and are expressed
in the molar concentration of angiotensin
produced per hour rather than in units, since
the conditions of assay differ somewhat from
those used elsewhere. They illustrate that
pseudorenin was found in all of the serums
tested and in amounts which are not related to
their renin content.
Discussion
Salivary Gland
Unitf/grain
FIGURE 7
Distribution of pseudorenin in the tissues of the rat.
A radiochemical method for the assay of
renin in human serum using a 14C-labeled
tetradecapeptide substrate has been under
development in this laboratory by one of us
Circulation Research, Vol. XXV, October 1969
NEW ANGIOTENSIN-FORMING ENZYME
461
TABLE 2
Renin and Pseudorenin Assays in Human Serum from Eight Subjects
Sample
Pooled serum
Normal
Normal
Normal
Normal
Normal
Hypertensive
Hypertensive
Benign hypertension
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(K. L.). During the course of this work, it was
found that more angiotensin was produced by
serum acting on the tetradecapeptide substrate than could be accounted for by the
renin content as determined by biological
methods. This finding led eventually to the
discovery of the new enzyme pseudorenin.
It was first thought that the new enzyme
was a degraded form, or perhaps a precursor,
of renin. Many experiments were performed
in an attempt to substantiate either of these
possibilities. All such attempts were unsuccessful, and there is no evidence that any
relationship exists. In fact, the pH optima of
the two enzymes are markedly different.
Further, they appear to be chemically distinct,
since they can be so easily separated on a
DEAE-cellulose column. The final argument
against any such relationship is the extremely
wide distribution of pseudorenin. Its activity
has been detected in every tissue or fluid thus
far examined. In contrast, only small amounts
of renin are found in extrarenal locations; and
except for the renin-like enzyme in the
submaxillary gland (10), it can be fairly said
that renin is a kidney enzyme and that it is
synthesized and stored in this organ in large
amounts (10, 11).
The new enzyme can be distinguished from
pepsin, which is also capable of producing
angiotensin I (12). The latter enzyme acts on
the natural protein substrate as it exists in
plasma, whereas pseudorenin does not. In
addition, angiotensin is an intermediate product of the hydrolytic action of pepsin and is
destroyed by longer incubations with the
Circulation Research, Vol. XXV,
October 1969
Pseudorenin
(mmoles/hr/ml)
Renin
(pmoles/hr/ml)
17.6
17.6
21.6
25.6
25.6
25.6
11.0
9.6
11.2
3.24
1.61
1.15
0.
1.44
1.15
26.2
60.0
7.5
enzyme (13). In contrast, incubation of
pseudorenin with the tetradecapeptide substrate may be continued without loss long
after the maximum biological activity is
attained. Chemical analysis of such a long
incubation showed the presence of only
angiotensin I and Leu-Val-Tyr-Ser. Thus
pseudorenin is a relatively specific enzyme
which hydrolyzes the Leu-Leu bond of the
tetradecapeptide substrate and differs from
pepsin, which has more general requirements
as to specificity.
Despite its widespread occurrence, it is
difficult to understand how pseudorenin can
actually function. It hydrolyzes the tetradecapeptide substrate very rapidly, but this
compound is not known to exist in the body. It
does not act on the natural substrate of the
plasma, and the presence of substrate in
tissues has not been shown. The finding that
pseudorenin does produce angiotensin I from
purified hog substrate A and the inhibition of
this reaction by serum is surprising; it suggests
that the reaction might proceed in vivo if a
mechanism existed for removing or otherwise
eliminating the inhibitory effect of serum from
the site of the reaction.
There remains the problem that pseudorenin reacts with greatest velocity at pH 4.5 to
5.0, which is far below the physiological
range. It is true that its activity against
substrate A was not detectable at pH 7.5,
although it should be noted that the experiment used human enzyme and hog substrate,
and that pure human substrate might well be
attacked at neutrality. It does, for example,
462
SKEGGS, LENTZ, KAHN, DORER, LEVINE
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attack the tetradecapeptide substrate at neutral pH. In fact, the amount of the enzyme in
human serum is so great that most of the
angiotensin produced by the incubation of
human serum with the tetradecapeptide substrate at pH 7.5 is due to the action of
pseudorenin rather than to that of renin.
Further, there is much more pseudorenin in
the tissues than in the plasma, so that its
extravascular action seems even more possible.
Finally, the enzyme may have an intracellular
locus of action, where the existing pH value is
not really known.
It would seem unlikely that an enzyme
would exist for which there was no substrate,
or that it would be found in a location where
it could not function. It seems more probable
that it does act, and that the product is
angiotensin I, which is liberated in the tissues.
Whether this decapeptide is then converted to
angiotensin II and what the function of either
of these peptides might be at the site of their
liberation are completely unknown.
References
1.
SKEGCS, L. T., JR., KAHN, J. R., LENTZ, K. E., AND
SKEGGS, L. T., JR., LENTZ, K. E., KAHN, J. R., AND
SHUMWAY, N. P.: Synthesis of a tetradecapeptide renin substrate. J. Exptl. Med. 108: 283,
1958.
5.
SKEGGS, L. T., LENTZ, K. E., KAHN, J. R., AND
HOCHSTRASSER, H.: Kinetics of the reaction of
renin with nine synthetic peptide substrates. J.
Exptl. Med. 128: 13, 1968.
6.
SKECCS, L. T., LENTZ, K. E., KAHN, J. R., AND
HOCHSTRASSER, H.: Studies on the preparation
and properties of renin. Circulation Res. 21
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7.
MARSHALL, G.
R.,
AND MERRIFIELD, R.
B.:
Synthesis of angiotensins by the solid-phase
method. Biochemistry 4: 2394, 1965.
8. Renin, in Renal Hypertension, edited by I. H.
Page and J. W. McCubbin. Chicago, Year
Book Medical Publishers, Inc., 1968, p. 19.
9.
COULD, A. B., SKECCS, L. T., AND KAHN, J.
R.:
Measurement of renin and substrate concentrations in human serum. Lab. Invest. 15: 1802,
1966.
10. Renin, in Renal Hypertension, edited by I. H.
Page and J. W. McCubbin. Chicago, Year
Book Medical Publishers, Inc., 1968, pp. 5658.
11.
GOULD, A. B., SKEGCS, L. T., JR., AND KAHN, J.
R.: Presence of renin activity in blood vessel
walls. J. Exptl. Med. 119: 389, 1964.
12.
FERNANDEZ, M.
T.
F.,
PALADINI, A. C ,
AND
DELIUS, A. E.: Isolation and identification of a
pepsitensin. Biochem. J. 97: 540, 1965.
SKEGCS, L. T., JR., LENTZ, K. E., HOCHSTRASSER,
H., AND KAHN, J. R.: Purification and partial
characterization of several forms of hog renin
substrate. J. Exptl. Med. 118: 73, 1963.
3.
4.
SKECCS, L. T., LENTZ, K. E., COULD, A. B.,
HOCHSTRASSER, H., AND KAHN, J. R.: Biochem-
istry and kinetics of the renin angiotensin
system. Federation Proc. 26: 42, 1967.
2.
SHUMWAY, N. P.: Preparation, purification and
amino acid sequence of a polypeptide renin
substrate. J. Exptl. Med. 106: 439, 1957.
13.
BRAUN-MENENDEZ, E., FASCIOLO, J. C , LELOIR,
L. F., MUNOZ, J. M., AND TAQULNI, A. C.
(eds.): Renal Hypertension. Springfield, 111.,
Charles C Thomas, 1946, p. 245.
Circulation Research, Vol. XXV, October 1969
Pseudorenin: A NEW ANGIOTENSIN-FORMING ENZYME
Leonard T. Skeggs, Kenneth E. Lentz, Joseph R. Kahn, Frederic E. Dorer and Melvin Levine
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Circ Res. 1969;25:451-462
doi: 10.1161/01.RES.25.4.451
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Copyright © 1969 American Heart Association, Inc. All rights reserved.
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