Download Hydrolysis of Bradykinin by Angiotensin

Hydrolysis of Bradykinin by Angiotensin-Converting Enzyme
By Frederic E. Dorer. Joseph R. Kahn, Kenneth E. Lentz, Melvin Levine, and Leonard T. Skeggs
ABSTRACT
Two dipeptides, phenylalanylarginine (Phe-Arg) and serylproline (Ser-Pro), are released sequentially from bradykinin by angiotensin-converting enzyme purified from hog
lungs; chloride increases the rate of release of both dipeptides. Using an automated
ninhydrin-reagent method, we studied the kinetics of bradykinin hydrolysis. The reaction
proceeded in the absence of chloride; however, the addition of chloride increased the rate
of hydrolysis by decreasing Km and increasing Vm . The Km values for bradykinin were 3.9
x 10~6M in the absence of chloride and 0.85 x 10~6M in the presence of 0.01M NaCl
(optimal concentration). Both of these Km values were well below the value of 30 x 10" 6 M
determined for angiotensin I at its optimal chloride concentration of 0.1M. Hydrolysis of
bradykinin had a pH optimum of 7 and was inhibited by low concentrations (10~ 6 M) of
ethylenediaminetetraacetic acid or the nonapeptide pyroglutamyl (Pyr)-Trp-ProArg-Pro-Gln-Ile-Pro-Pro. It is concluded that one enzyme, acting as a dipeptidyl
carboxypeptidase, catalyzes both the conversion of angiotensin I to angiotensin II and the
hydrolysis of bradykinin.
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KEY WORDS
phenylalanylarginine
enzyme kinetics
dipeptidyl carboxypeptidase
chloride activation
serylproline
hog lung
Bothrops jararaca nonapeptide
• Extracts of mammalian lung contain an enzyme
or enzymes capable of converting angiotensin I to
angiotensin II and of destroying the biological
activity of bradykinin (1). (Hydrolysis of any
peptide bond in bradykinin [Arg-Pro-Pro-GlyPhe-Ser-Pro-Phe-Arg] abolishes biological activity
[2].) Recently, an enzyme purified from lung has
been shown to possess both of these activities (3).
Bradykinin is a competitive inhibitor of angiotensin conversion (4), implying a common enzyme.
However, the hydrolysis of bradykinin by angiotensin-converting enzyme preparations is reported to
be independent of the presence of chloride; this
finding leads to speculation that the enzyme activities must differ in some way (4, 5). The availability
of a purified enzyme preparation (6) and the
development of a quantitative chemical assay for
measuring the hydrolysis of bradykinin enabled us
to investigate whether bradykinin and angiotensin
are hydrolyzed by the same enzyme and whether
chloride participates in the bradykininase reaction.
Methods
Angiotensin I, hippuryl (Hip)-Gly-Gly, and His-Leu
were synthesized as described previously (6, 7). Bradykinin was purchased from Schwarz/Mann, and N-2From the Department of Medicine and Surgery, Veterans
Administration Hospital, Cleveland, Ohio, and the Departments of Biochemistry and Pathology, Case Western Reserve
University, Cleveland, Ohio 44106.
Received June 15, 1973. Accepted for publication March 19,
1974.
824
hydroxyethylpiperazine - N' - 2 - ethanesulfonic acid
(HEPES) was purchased from Calbiochem. The dipeptides, serylproline (Ser-Pro), phenylalanylarginine (PheArg), and Gly-Phe, were purchased from Fox Chemical
Company. The concentrations of standard solutions of
the peptides were determined by amino acid analysis
following acid hydrolysis. The synthetic peptide pyroglutamyl (Pyr)-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro was a
gift.1 The angiotensin-converting enzyme purified from
hog lung was obtained from fraction G or fraction H as
described previously (6). Fraction H has been shown to
be homogeneous by disk gel electrophoresis at pH 9.5 (6).
These two fractions are identical with regard to relative
rates of bradykinin, angiotensin I, and Hip-Gly-Gly
hydrolysis, chloride activation, and similarity of dipeptide formed from bradykinin.
Enzyme assays were performed by a modification of
the automated method of Skeggs et al. (8) in which the
rate of peptide bond hydrolysis is measured continuously
by the ninhydrin reaction. Increased sensitivity was
obtained by using the Technicon AutoAnalyzer II colorimeter, which permitted a twenty-fold electrical range
expansion so that 6 x 10" 6 M leucine gave a full-scale
reading. The incubation mixtures (final volume 5 ml)
contained enzyme in 0.025M HEPES buffer (pH 7.5) with
0.04% Brij-35 (a synthetic detergent [7]) and NaCl at the
indicated concentration. After a 10-minute incubation at
37°C, the substrate was added, and the mixture was
sampled continuously for 8 minutes at 37°C. Enzyme
velocities were calculated directly from the slopes of the
recordings and were expressed as nanomoles of dipeptide
formed per minute in the 5-ml incubation mixture. The
analytical system was calibrated with His-Leu when
angiotensin I was the substrate, with Gly-Gly when
1
Generously supplied by Dr. J. W. Ryan. University of
Miami School of Medicine.
Circulation Research. Vol. XXXIV. June 1974
825
BRADYKININ HYDROLYSIS BY CONVERTING ENZYME
Hip-Gly-Gly was the substrate, and with Ser-Pro or
Phe-Arg (identical ninhydrin color values. 83^ of leucine) when bradykinin was the substrate.
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Results
The automated enzyme assay method used in
this study for bradykinin hydrolysis yielded velocities that were linear with time during the 8-minute
sampling period and enzyme concentrations that
were in the range of 0.1 to 1.0 Mg/ml. At pH 7.5, the
Km and Vm values for bradykinin hydrolysis (Fig.
1) were 3.9 x 10' 6 M and 0.81 /umoles/min mg~'
protein, respectively, in the absence of chloride,
and 0.85 x 10"6M and 1.4 ^moles/min mg"' protein
in the presence of 0.01M NaCl. Similarly, the Km
and Vm values for angiotensin I hydrolysis were 30
x 10" 6 M and 2.1 /umoles/min mg" 1 protein, respectively, in the presence of 0.1M NaCl.
The relationship between bradykinin hydrolysis
4.5-,
and chloride concentration is shown in Figure 2.
The optimal chloride concentration is about onetenth of that previously found for the angiotensinconversion reaction (phosphate buffer, pH 8) (6).
The observed velocity in the absence of added
chloride did not appear to result from the presence
of trace amounts of chloride in the reagents,
because there was no hydrolysis of Hip-Gly-Gly, a
reaction which absolutely requires chloride (6)
under these conditions. Also, bradykinin that had
been treated by chromatography on BioGel P-2 or
with an ion-exchange resin (AG 2-X8 acetate) to
remove any traces of chloride was still hydrolyzed
at the same rate in the absence of added NaCl.
The pH-activity relationships for bradykinin and
angiotensin I hydrolysis are shown in Figure 3. The
shapes of the curves for bradykinin hydrolysis in
the presence and the absence of chloride are
similar. Figure 4 shows the time course of bradykinin hydrolysis with and without chloride. Chloride
increased the rate of hydrolysis without changing
the final value attained.
The hydrolysis of bradykinin (4 x 10" 6 M) both in
the presence and the absence of NaCl was inhibited
more than 50% by 10" 6M ethylenediaminetetraacetic acid (EDTA) or 10" 6M Pyr-Trp-Pro-Arg3.0
0.5
1.0
1.5
2.0
Double reciprocal plot of bradykinin hydrolysis showing the
effect of chloride on Km and Vm . Velocities are expressed as
nmoles dipeptide formed/min. and substrate concentrations are
expressed as nmoleslml. Assays were carried out as described in
the text in the absence of NaCl with 2.5 ng of enzyme {solid
circles) and 5.0 ng of enzyme {triangles) and in the presence of
0.01 M NaCl with 1.0 fig of enzyme (crosses) and 2.5 fig of enzyme (open circles).
Circulation Research. Vol. XXXIV. June 1974
10"
10
10"
10"
NaCl concentration (M)
FIGURE 2
Bradykinin hydrolysis as a function of NaCl concentration.
Assays were carried out as described in the text using 2.5 ng of
enzyme and 20 nmoles of bradykinin.
826
DORER. KAHN. LENTZ, LEVINE, SKEGGS
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FIGURE 3
Bradykinin hydrolysis and angiotensm-converting enzyme activity as a function of pH. Assays were carried out with 2-5 ng of
enzyme as described in the text except that HEPES buffers of
varying pH were used. The pH values were measured at 25°C at
the end of the incubations. Circles - hydrolysis of 20 nmoles of
bradvkinin in the presence of 0.01M NaCl. triangles = hydrolysis
of 20 nrrwles of bradykinin in the absence of NaCl, and crosses hydrolysis of 200 nmoles of angiotensm 1 in the presence of 0.1 M
NaCl.
Pro-Gln-Ile-Pro-Pro, a bradykinin-potentiating
peptide found in Bothrops jararaca venom (9).
The demonstration by chemical methods of the
hydrolysis of bradykinin by angiotensin-converting
enzyme was confirmed by the loss of biological
activity of bradykinin. Incubation for 1 hour with
an excess of enzyme (with or without NaCl) completely abolished the depressor effect of bradykinin
(1 nmole, iv) injected into an anesthetized rat (10).
To determine which peptide bonds were hydrolyzed, bradykinin (260 nmoles) was incubated for 1
hour with an excess of converting enzyme in a
volume of 0.7 ml at pH 7.5; this procedure was
followed by identification of products on the amino-acid analyzer. The results were the same with
and without chloride; two peaks were observed that
coincided with Ser-Pro (240 nmoles) and Phe-Arg
(280 nmoles). No free arginine or Gly-Phe was
found.
The effect of NaCl on the initial rate of release of
Phe-Arg and Ser-Pro is shown in Figure 5. The
bradykinin concentration used in this experiment
2.4-
2.0-
1.6-
1.2-
o.e0.4-
10
20
30
40
Time (min)
50
60
FIGURE 6
0
20
40
60
Time (min)
FIGURE 4
Time course of the hydrolysis of bradykinin. Twenty nmoles of
bradykinin were incubated with 5 ng of enzyme as described in
the text except that sampling was done discontinuously at the
indicated times from an incubation volume of 10 ml. Circles
indicate the absence of NaCl, and crosses indicate the presence
of 0.01M NaCl.
Release of Phe-Arg and Ser-Pro from bradykinin. Bradykinii
(100 nmoles) was incubated with 1 fig of enzyme at pH 7.5 in i
total volume of 25 ml in the presence and the absence of NaC
for time intervals up to 1 how. The reaction was stopped b
boiling, and the entire incubation mixture was pumped onto th
column of the amino-acid analyzer, modified (11) with
gradient to elute Phe-Arg. Solid circles - Phe-Arg formed in th
presence of O.OlM NaCl, triangles - Phe-Arg formed in th
absence of NaCl. open circles - Ser-Pro formed in the presenc
of 0.01M NaCl, and crosses =• Ser-Pro formed in the absence <
NaCl.
Circulation Ratarth, Vol. XXXIV, June IS,
827
BRADYKININ HYDROLYSIS BY CONVERTING ENZYME
was 4 x 10 6M, the same as that used in the
experiments considered in Figures 2 and 3 and
twice that in the experiments considered in Figure
4. The rate of release of both dipeptides depended
on chloride (Fig. 5); in the case of Phe-Arg, the
stimulation by chloride was about 2.5-fold.
hydrolysis of bradykinin has been presented. This
conclusion is compatible with the dipeptidyl carboxypeptidase type of specificity previously described for this enzyme (3).
Discussion
1. BAKHLE, Y.S.: Conversion of angiotensin I to angiotensin II
by cell-free extracts of dog lung. Nature (Lond)
220:919-921, 1968.
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Most of the data presented in this paper represent a mixed reaction, i.e., the actual quantity
measured was the sum of Phe-Arg and Ser-Pro.
Nevertheless, the initial mixed rate, at least under
the experimental conditions of Figure 5, appears to
represent the initial reaction, which was the release
of Phe-Arg.
Evidence that chloride participates in the hydrolysis of bradykinin was found. Although it is not
an absolute requirement, chloride does affect both
Km and Vm . The data presented in Figure 5 show
that chloride is required for maximal rate of release
of both dipeptides. Since the bradykinin molecule
becomes inactive when the Phe-Arg moiety is
removed, the rate of Phe-Arg release should parallel the loss of biological activity (bradykininase
activity). Thus, it is difficult to understand why
the chloride effect has not been observed by other
investigators (4, 5) using biological assays.
It is not known whether the pulmonary converting enzyme-bradykininase described in this paper
is the enzyme responsible for bradykinin inactivation under physiological conditions in the intact
animal. Bradykinin is hydrolyzed at several bonds
during perfusion through the blood-free rat lung
(2). However, the inactivation of bradykinin in the
pulmonary circulation is inhibited by Bothrops
jararaca peptides (5, 12).
Evidence that one enzyme, which requires chloride for maximal activity, catalyzes both the conversion of angiotensin I to angiotensin II and the
References
2. RYAN, J.W., ROBLERO. J., AND STEWART, J.M.: Inactivation
of bradykinin in rat lung. Adv Exp Med Biol 8:263-271,
1970.
3. IGIC, R., ERDOS, E.G., YEH, H.S.J., SORRELLS, K., AND
NAKAJIMA, T.: Angiotensin I converting enzyme of the
lung. Circ Res 31(suppl. II):II-51-61. 1972.
4. SANDER, G.E., WEST, D.W., AND HUGGINS, C.G.: Peptide
inhibitors of pulmonary angiotensin I converting enzyme.
Biochim Biophys Acta 242:662-667, 1971.
5. ALABASTER, V.A., AND BAKHLE, Y.S.: Converting enzyme and
bradykininase in the lung. Circ Res 31(suppl. II): 11-72-81,
1972.
6. DORER, F.E., KAHN, J.R., LENTZ, K.E., LEVINE, M., AND
SKEGGS, L.T.: Purification and properties of angiotensinconverting enzyme from hog lung. Circ Res 31:356-366,
1972.
7. DORER, F.E., SKEGGS, L.T., KAHN, J.R., LENTZ, K.E., AND
LEVINE, M.: Angiotensin converting enzyme: Method of
assay and partial purification. Anal Biochem 33:102-113,
1970.
8. SKEGGS, L.T., LENTZ, K.E., KAHN, J.R., AND HOCHSTRASSER,
H.: Kinetics of the reaction of renin with nine synthetic
peptide substrates. J Exp Med 128:13-34, 1968.
9. FERREIRA, S.H., BARTELT, D.C., AND GREENE, L.J.: Isolation
of bradykinin-potentiating peptides from Bothrops
jararaca venom. Biochemistry 9:2583-2593, 1970.
10. SKEGCS, L.T., JR., KAHN, J.R., AND MARSH, W.H.: Method
of assaying small amounts of Hypertensin. Lab IrTvest
2:109-114, 1953.
11. MITCHELL, A.R., AND ROESKE, R.W.: Chromatography of
im-benzyl-L-histidine. J Chromatogr 43:266-267, 1969.
12. GREENE, L.J., CAMARCO, A.CM., KRIECER, E.M., STEWART,
J.M., AND FERREIRA, S.H.: Inhibition of the conversion of
angiotensin I to II and potentiation of bradykinin by
small peptides present in Bothrops jararaca venom. Circ
Res 31(suppl. II):II-62-71, 1972.
Correction
Vol. 34, p 66
Table 3, line five, should read
TABLE 3
Author
Present study
Circulation Research. Vol. XXXIV. June 1974
Reference
Compliance
(ml/mm Hg kg '
body wt)
Animal
2.3
Man
Hydrolysis of Bradykinin by Angiotensin-Converting Enzyme
FREDERIC E. DORER, JOSEPH R. KAHN, KENNETH E. LENTZ, MELVIN LEVINE and
LEONARD T. SKEGGS
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Circ Res. 1974;34:824-827
doi: 10.1161/01.RES.34.6.824
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