Kinetics of the Inhibitory Effect of Pepstatin on the Reaction of
Hog Renin with Rat Plasma Substrate
By Helmut Orth, Eberhard Hackenthal, Jeffrey Lazar, Ulrike Miksche, and Franz Gross
ABSTRACT
The reaction between hog renin and rat angiotensinogen followed Michaelis-Menten
kinetics. A Km value of 1,109 ng angiotensin/ml was determined for the rate of angiotensin
formation at pH 7.4 and 37°C. The pH optimum for the reaction of hog renin with rat
angiotensinogen was between 6.8 and 7.0. Pepstatin inhibited the reaction in a
noncompetitive way. The maximum inhibitory effect occurred at approximately pH 6; at
values above pH 8.0, pepstatin had no inhibitory effect. The effect of pepstatin was
reversible, since renin activity could be restored by 24 hours of dialysis against distilled
water. A 50% inhibition of the enzymatic activity of 0.01 dog units of hog renin was
obtained with 0.45 ng pepstatin/ml.
KEY WORDS
enzyme inhibitor
hog renin
rat angiotensinogen
enzyme kinetics
angiotensin
noncompetitive inhibition
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• Pepstatin, a pentapeptide isolated from the
culture filtrate of actinomycetes strains (1) and
initially characterized as an inhibitor of pepsin and
other acid proteases (2, 3), has also been shown to
inhibit the reaction of hog renin with rat plasma
s\ibstrate at a neutral pH. The interaction of renin
with heterologous plasma substrate has been demonstrated both in vitro (4) and in vivo in rats (5, 6).
Although various authors have confirmed the inhibitory effect of pepstatin on renin, no extensive
studies on the kinetics of this reaction have been
published. Recent studies (6, 7) have claimed that
competitive antagonism occurs, but evidence for
such a claim is not convincing. Preliminary studies
in our laboratory have indicated a noncompetitive
type of inhibition. Therefore, the present investigation attempted to elucidate the mechanism underlying the inhibitory effect of pepstatin on the release of angiotensin from rat plasma substrate by
hog renin.
Methods
RENIN
Solutions of hog renin (0.1 dog units/ml) were prepared in 0.067M Soerensen phosphate buffer, pH 7.4, and
human albumin (final concentration 1 mg/ml) was
added to the solutions, which were then stored at -25°C.
of rats subjected to a bilateral nephrectomy 24 hours
earlier. The lyophilized substrate preparation contained
an amount of angiotensinogen equivalent to 100 ng
angiotensin/mg protein.
PEPSTATIN
Pepstatin1 was dissolved in a phosphate-buffered (pH
8) 0.9% saline solution in a concentration of 4 Mg/ml and
stored at -25°C. Mixtures of enzyme, substrate, and
inhibitor were incubated at pH 7.4 and 37°C for 10
minutes. For determinations of the pH optimum, the
incubation period was extended to 20 minutes to obtain
more distinct differences in the amounts of angiotensin
formed at various values of pH. When the substrate
concentration was varied, the duration of incubation was
reduced to 5 minutes in the experiments with a low
concentration of substrate to keep substrate consumption below 10% of the initial amount. The incubate
contained an amount of angiotensinogen equivalent to
4,500 ng angiotensin/ml solution. Each milliliter of
solution contained 0.067M phosphate buffer, 0.01M Na,
ethylenediaminetetraacetate (EDTA), 0.02% neomycin,
0.1 ml of hog renin (0.01 dog units) solution, and 0.1 ml of
pepstatin (0.4 ng) or, in control experiments, 0.1 ml of
0.9% saline. At the end of the incubation period, the
reaction was stopped by the addition of 0.2 ml of IN
hydrochloric acid followed by boiling in a water bath for
5 minutes. Then 0.7 ml of 0.067M Na,HP0 4 was added to
correct the pH, and the samples were frozen and stored
at -25 C C. Details of the method have been described
previously (10).
DETERMINATION OF ANGIOTENSIN
SUBSTRATE
The substrate was prepared according to the method
of Haas et al. (8) and Boucher et al. (9) from the plasma
From the Department of Pharmacology, University of Heidelberg, 6900 Heidelberg, Germany.
This study was supported in part by the German Research
Foundation within the SFB 90, CardiovasculSres System.
Received November 2, 1973. Accepted for publication March
28, 1974.
62
To ascertain the Michaelis constant (Km) and the
inhibitor constant (K|), the concentration of angiotensin
in the samples was estimated by a radioimmunoassay
specific for angiotensin I, which was similar to that
described by Haber et al. (11). In control experiments,
care was taken to ensure that only angiotensin I was
1
Kindly supplied by Professor H. Umezawa, Institute of
Microbial Chemistry, Tokyo, Japan.
Circulation Racarth. Vol. 35, July 1974
PEPSTATIN KINETICS
53
formed. In all other experiments, angiotensin concentrations were measured by bioassay in the rat (10).
ng
Ang
Results
pH DEPENDENCE
For the reaction between hog renin and rat
plasma substrate, the pH optimum was between
6.8 and 7.0. At a pH above 8.0, the activity of hog
renin rapidly declined (Fig. 1). The inhibitory
effect of pepstatin on the hog renin-rat substrate
reaction was most marked in the acid pH range.
At a pH above 6.8 pepstatin lost activity, and at pH
values greater than 8 no inhibitory effect was
demonstrated.
2000
BINDINO OF PEPSTATIN TO ANOIOTENSINOGEN
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Since binding of the pepstatin molecule to the
substrate might interfere with the enzyme-substrate interaction, the question of whether rat
substrate in the presence of pepstatin can be converted quantitatively to angiotensin by an excess of
hog renin was raised. Various amounts of substrate
(434-3,472 ng angiotensin/ml) with and without
pepstatin were incubated with 1 dog unit of hog
renin (Fig. 2). The results of this experiment
showed that pepstatin was not irreversibly bound
to an active site of the substrate, since the amount
of angiotensin cleaved from the substrate in the
presence of pepstatin was the same as that cleaved
in the absence of pepstatin.
BINDING OF PEPSTATIN TO RENIN
To determine whether the binding of pepstatin
to renin was reversible, a mixture of renin (0.1 dog
ng
Ang
o o
o
o
300 •
0
°t'o
o
o
200 o
•
o
o
•
•
•
m
888
1736
3*72
ng Ang/mL
FIGURE 2
Incubation of hog renin (1 dog unit) with increasing amounts of
rat plasma substrate without (open circles) and with (solid
circles) the addition of 05 ng pepstatinlml. Ordinate - angiotensin released after a 10-minute incubation, and abscissa substrate concentration (equivalent to ng angiotensin/ml).
units/ml) and pepstatin (4 jig/ml) was dialyzed
against distilled water for 24 hours at 0°C. The
renin activity in the dialyzed, lyophilized, and
reconstituted sample was compared with that in a
similarly treated control sample which contained
no pepstatin and also with that in nondialyzed
control samples. The renin activity in the sample
containing pepstatin was 82 ng angiotensin formed
before and 135 ng angiotensin formed after dialysis,
whereas the renin activity in the sample without
pepstatin was 134 ng angiotensin formed before
and 132 ng angiotensin formed after dialysis.
Therefore, the inhibition of the renin-substrate
reaction was completely reversible.
DETERMINATION OF K, A N D K .
o
100 •
434
» o
•
10
pH
FIGURE 1
Reaction between hog renin (0.01 dog units/ml) and rat angiotensinogen (4,500 ng/ml) at various values of pH (open circles)
and the effect of pepstatin (0.4 fig/ml) on the renin-substrate
reaction (solid circles). At pH 7.4 the activity of renin with the
addition of pepstatin was 47% of the control without pepstatin.
Ordinate = angiotensin released after a 20-minute incubation,
and abscissa - pH values.
Circulation Ratarth. Vol. 35. July 1974
The pepstatin concentration that produced a
50% inhibition of the activity of hog renin (0.01 dog
units) was ascertained by the method of Dixon and
Webb (12). By using two substrate concentrations
(1,060 ng angiotensin/ml and 4,690 ng angiotensin/
ml) and varying the pepstatin concentration between 0 and 0.8 ngim\, a K, value of 0.455 fig
pepstatin/ml was obtained (Fig. 3). The finding
that the two lines intersected the abscissa at
virtually the same point indicated a noncompetitive inhibition.
To verify this finding and to further characterize
the type of inhibition, the reaction between hog
renin and rat substrate was studied at various
substrate concentrations between 222 and 4,440 ng
angiotensin/ml with and without the addition of
54
ORTH. HACKENTHAL, LAZAR. MIKSCHE, GROSS
200
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-K.
-
Pep.
0,2
0,6
0,8
FIGURE 3
Determination of Ki.St = substrate equivalent to 1,060 ng
angiotensin/ml, S, - substrate equivalent to 4,690 ng angiotensin/ml, ordinate - reciprocal plot of angiotensin produced
by 0.01 dog units of hog renin (ng" x 10"), and abscissa concentration of pepstatin big/m[). Each point represents
the mean of three radioimmunological determinations of
duplicate incubations.
pepstatin (0.4 ug/m\). The values measured for
angiotensin were plotted by the Lineweaver-Burke
method (12). A Km of 1,109 ng angiotensin/ml was
found at pH 7.4 and 37°C, and the maximum rate
of angiotensin formation (Vm,x) was 17.4 ng angiotensin/min with 0.01 dog units of hog renin. When
pepstatin (0.4 fig/ml) was added, V mai fell to 8.4 ng
angiotensin/min. Hence, by means of the Lineweaver-Burke plot, a noncompetitive type of inhibition
was also observed. Pepstatin reduced the Km value
from 1,109 ng angiotensin/ml to 804 ng angiotensin/ml (Fig. 4).
Discussion
The results obtained in this study clearly indicate that pepstatin inhibits the reaction between
hog renin and rat plasma substrate in a noncompetitive way. This finding contrasts with the observations reported by other investigators (6, 7), who
have proposed a competitive type of inhibition. An
interpretation of these divergent findings is not
possible, since no detailed information about the
method used or the results obtained is available.
Originally, pepstatin was described as a noncompetitive inhibitor of pepsin (1). Kunimoto et al. (2)
then found a competitive inhibition of pepsin for
diacetylpepstatin when a special substrate,
Af-acetyl-L-phenylalanyl-L-diiodotyrosine, was
used. These authors (2) concluded that, under
certain conditions, pepstatin can also act as a
competitive inhibitor of pepsin. Our data do not
permit conclusions as to whether the renin-substrate reaction is inhibited in a noncompetitive way
under all conditions, but recently Bath and Gregerman have found a noncompetitive inhibition of the
reaction between human renin and a polymeric
tridecapeptide substrate (13) by pepstatin (personal communication).
Whether the differences in the Km values that
were calculated with and without the addition of
pepstatin are due to an increase in the affinity of
the enzyme-inhibitor complex for the substrate or
merely reflect variations in measurement is not
clear. Further studies are necessary to answer this
question. The fact that pepstatin can be completely removed from a mixture with renin by
dialysis demonstrates that the binding of pepstatin
to renin is reversible. The same phenomenon has
been shown for the binding of pepstatin to pepsin
(2).
300
200
•
100 -
100
200
FIOURE-4
Lineweaver-Burke plot of an incubation of 0.01 dog units of hog
renin with various amounts of rat angiotensinogen without
{open circles) and with (solid circles) the addition of pepstatin
(0.4 ng/ml). Ordinate = ng~' angiotensin x 10~', and abscissa
- (ng angiotensin/ml)'' x 10~'. Each point represents the
mean of three radioimmunological determinations of duplicate
incubations.
Curulcaion Raeareh. Vol. 35. July 1974
55
PEPSTATIN KINETICS
The observation that the extent of inhibition by
pepstatin is pH dependent corresponds to former
data obtained with rat renin and homologous
substrate (10). This pH effect probably reflects
differences in the degree of dissociation at the sites
of pepstatin that are responsible for the binding to
renin.
H, MATSUZAKI M,
HAMADA M, TAKEUCHI T: Pepstatin, a new pepsin inhibitor produced by actinomycetes. J Antibiot (Tokyo)
23:259-262, 1970
2. KUNIMOTO
S,
AOYAGI T,
MORISHIMA
H, TAKEUCHI
T,
UMEZAWA H: Mechanism of inhibition of pepsin by
pepstatin. J Antibiot (Tokyo) 25:251-255, 1972
3. AOVAGI T,
KUNIMOTO
S,
MORISHIMA
H, TAKEUCHI
T,
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UMEZAWA H: Effect of pepstatin on acid proteases. J
Antibiot (Tokyo) 24:687-694, 1971
4. GROSS F, LAZARJ, ORTHH: Inhibition of the renin-angioten-
sinogen reaction by pepstatin. Science 175:656, 1972
5. LAZAR J, ORTH H, MOHRINC J, GROSS F: Effects of the renin
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nephrectomized rats. Naunyn Schmiedebergs Arch Pharmacol 275:114-118, 1972
Circulation Rntwxh. Vol. 35, July 1374
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Biochem
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Kinetics of the Inhibitory Effect of Pepstatin on the Reaction of Hog Renin with Rat Plasma
Substrate
HELMUT ORTH, EBERHARD HACKENTHAL, JEFFREY LAZAR, ULRIKE MIKSCHE and
FRANZ GROSS
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Circ Res. 1974;35:52-55
doi: 10.1161/01.RES.35.1.52
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