Download Endooligopeptidase A Activity in Rabbit Heart

Peptides, Vol. 9, pp. 945--955. ©PergamonPress plc, 1988. Printedin the U.S.A.
0196-9781/88$3.00 + .00
Endooligopeptidase A Activity in
Rabbit Heart: Generation of Enkephalin
From Enkephalin Containing Peptides
MARIA APARECIDA CICILINI, MARIA JOSE FONSECA RIBEIRO,*
EDUARDO BRANDT DE OLIVEIRA,$ RENATO ARRUDA MORTARA't
AND ANTONIO CARLOS MARTINS DE CAMARGO§ 1
Department of Physiological Sciences, Universidade Federal do Espirito Santo, ES, Brazil
*Department of Toxicology, Faculdade de Ci~ncias FarmacPuticas de Ribeirao Preto
Universidade de Sao Paulo, SP, Brazil
tDepartment of Parasitology Escola Paulista de Medicina, Sao Paulo, SP, Brazil
"~Department of Immunology, and §Department of Pharmacology, lnstituto de Ci~ncias Biom~dicas
Universidade de Sao Paulo, Caixa Postal 4365, 01000 Sao Paulo, SP, Brazil
R e c e i v e d 13 J a n u a r y 1988
CICILINI, M. A., M. J. F. RIBEIRO, E. B. DE OLIVEIRA, R. A. MORTARA AND A. C. M. DE CAMARGO.
Endoo/igopeptidase A activity in rabbit heart: Generationof enkepha/infrom enkephalin containingpeptides. PEPTIDES
9(5) 945--955, 1988.--Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in
molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel
filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described
rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met5] enkephalin and
[LeuS]enkephalinfrom small enkephalin containingpeptides. They also hydrolyze the PheS-Ser5 bond of bradykinin and the
Arg~-Arg9 bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When
electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of
Mr=73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by
immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr=72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulinreacts
with a polypeptide corresponding to Mr=50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are
activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating
that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A.
Heart endooligopeptidase A
Enkephalin-generating enzyme from heart
BIOLOGICALLY active peptides are processed by limited
proteolysis of larger precursor proteins known as prohormones (19). Observation of the amino acid sequence of
the pro-hormones reveals that the structure of biologically
active peptides within the precursor protein are, with few
exceptions, flanked by paired basic residues suggesting a
processing mechanism involving a proteolytic cleavage at
this site (34). Such putative processing signals are present in
pro-enkephalin (22) and pro-dynorphin (24) as well as in several naturally occurring enkephalin containing peptides derived from both precursors (37). Recently, enkephalins and a
number of opioid peptides derived from pro-enkephalin and
pro-dynorphin have been found in mammalian heart (23,39).
To explain the presence of these opioid peptides in heart
tissue an enkephalin generating system might also be present
within cardiac cells. Among the enzymes that may participate on the formation of enkephalin is endooligopeptidase A
(11,12). This enzyme isolated from the 25,000 g supernatant
fraction of nervous tissue with Mr=71,000, is a cysteinyl
endopeptidase with pH optimum of 7.2 and an isoelectric
point of 5.2 (8, 13, 29). Characteristically, endooligopeptidase A is only effective on small polypeptides (9) with
specificty towards the Phee-Ser~ bond of bradykinin (8) the
ArgS-Arga bond of neurotensin (10) and the MetS-Arg6 of
BAM-12P (11). More recently we reported on the ability of
endooligopeptidase A to form enkephalins from a range of
naturally occurring enkephalin containing peptides derived
from both pro-enkephalin and pro-dynorphin (12). Enkephalin containing peptides ranging from 8 to 13 amino acids in
size were shown to be good substrates for the enzyme, with
1Requests for reprints should be addressed to A. C. M. de Camargo.
945
946
CICILINI ET AL.
ABBREVIATIONS
Enk(Leu)
Enk(Met)
BAM-12P
BAM-22P
NT
EDTA
Tris
DFP
U
HPLC
SDS-PAGE
Mr
[LeuS]enkephalin
[MetS]enkephalin
Bovine adrenal medulla dodecapeptide
Bovine adrenal medulla docosapeptide
Neurotensin
Ethylenediamine-tetraacetic acid
Tris (hydroxymethyl)aminomethane
Diisopropyl fluorphosphate
Units of enzyme activity
High performance liquid chromatography
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
Relative molecular mass (molecular weight)
enkephalin being the only product released from precursors,
where this peptide is immediately followed by a pair of basic
residues. Following the observation that heart is among the
richest sources of endooligopeptidase A like enzyme (16) we
report here on the isolation and characterization of two
molecular weight forms of this enzyme in rabbit heart.
EXPERIMENTALPROCEDURES
Rabbit hearts were purchased from Granja Seleta (Itu,
Sao Paulo, Brazil) and stored at -20°C. Synthetic peptides
enk(Leu), enk(Met), BAM-12P, neurotensin and bradykinin
were from Cambridge Research Biochemicals; dynorphin
A,_8, dynorphin B, dynorphin A,-~7, neoendorphin (alpha),
neoendorphin (beta), BAM-22P, were from Peninsula Laboratories Ltd.; <Glul-LeuZ-Tyr3-Glu4-Asn3-Lysn-ProT-Arg8
(neurotensin, 8) and Arg.~-Pro'°-Tyr"-Iler'-Leu'3 (neurotensin~_,:0 were obtained by incubation of neurotensin with
rabbit brain endooligopeptidase A followed by purification
over HPLC (10); Argl-Pro2-Pro3-Gly4-Phe 5 (bradykinin,j
and Ser"-ProT-Phe~-Arg~ (bradykinin6_~) were synthesized by
the solid-phase method and purified by counter-current distribution and ion-exchange chromatography by Professor A.
C. M. Paiva and L. Juliano, Escola Paulista de Medicina,
Sao Paulo, Brazil. Horseradish peroxidase immunoglobulin
conjugates were from Dakko Corporation, Santa Barbara,
CA. Bovine serum albumin, Aldolase, Blue Dextran 2000,
Ribonuclease, Ovalbumin, Chymotrypsinogen and Sephacryl S-200 were from Pharmacia Fine Chemicals Inc. Diothiothreitol, aminopeptidase M, leupeptin, 5,5',-dithiobis (2nitrobenzoic) acid, sodium ethylenediamine tetraacetic acid,
p-OH-mercuribenzoate and diisopropyl fluorphosphate were
products of Sigma Chemical Co.; N-[1-(RS)-carboxy-2phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and alpha-Nbenzoyl-Gly-Ala-Ala-Phe-p-aminobenzoatewere a generous
gift of Dr. Marian Orlowski to O. Toffoletto and J. Rossier;
Acrylamide N'-methylene-bis-acrylamide and acetonitrile
were from Fluka AG, Buchs SG, Switzerland; DEAE-cellulose
(Whatman DE-52) was obtained from Reeve Angel (London,
United Kingdom). Aminex A-5 was from Bio-Rad Laboratories, Richmond, CA. Agarose was purchased from BDH
Chemicals (Poole, United Kingdom). W-3 ion-exchange resin
was from Beckman Instruments Co. and amino acid analyzer
reagents were purchased from Pierce Chemical Co., Rockford,
IL. Total cell extracts of epimastigote forms of Trypanosoma
cruzi were used as a crude source of tubulin (38). Anti-veal
skeletal muscle actin monospecific antibodies raised in rabbits
were kindly provided by S. Avrameas, from Institute Pasteur,
Paris. The monoclonal antibody against actin was purchased
from Amersham, England.
Enzyme Unit (U)
This is defined as the amount of enzyme that hydrolyzes
1.0 ~mol of bradykinin per min at 37°C in 50 mM Tris-HCl,
pH 7.5, containing 0.35 mM dithiothreitol and 10/zM substrate.
Enzyme Extraction
The enzyme was prepared at 4°C from 30 rabbit hearts
homogenized in 1:3 weight:volume of l0 mM of Tris-HCl
buffer pH 7.5, containing 0.25 M sucrose, for l rain at top
speed in a 2 l Waring blender. The homogenate was centrifuged at 25,000×g for 60 rain and the precipitate discarded.
Acid Precipitation
The pH of the supernatant fraction of rabbit heart extract
was adjusted to 5.0 by dropwise addition of 0.5 M acetic acid
and maintained at 4°C for 15 min, the precipitate being discarded after centrifugation. The pH of the supernatant was
then adjusted to 7.5 by addition of 0.5 M NaOH.
DEAE-Celhdose Chromatography
The DEAE-cellulose column (2.5×70 cm) was equilibrated with 50 mM Tris-HC1 buffer, pH 7.5, containing 30 mM
NaC1. After sample application, the column was developed
by stepwise elution with increasing NaCI concentrations.
The column was operated at 220 mlhar at 4°C, and fractions
of 9 ml were collected. Kininase activity (bradykinin inactivating activity) was determined by bioassay with isolated
guinea pig ileum (7). The protein from the effluent was monitored by UV absorbance at 280 nm.
Gel-Filtration on Sephacryl S-200
The column (2.5 × 110 cm) was equilibrated and developed
at 4°C with 50 mM Tris-HCl buffer. The apparent Mr of the
enzyme was estimated by the method of Andrews (2), using
ribonuclease, chymotrypsinogen, bovine serum albumin and
aldolase as Mr standards.
Preparative Disc Electrophoresis on Polyacrylamide Gels
Fractions DE-I, S-I, S-II and S-III (Figs. 1 and 2) were
concentrated by ultrafiltration under reduced pressure and
dialyzed at 4°C overnight against 50 mM Tris-HC1, pH 6.7,
containing 20% glycerol. Polyacrylamide gel electrophoresis
was carried out at 5°C, according to Ornstein-Davis method
(18,32) on 6% acrylamide gels with bis-acrylamide: acrylarnide ratio 1:40. At the end of the run the gels were either
stained for protein with Coomassie brilliant blue or assayed
for enzyme activity as follows: the gel was out transversely
into 40 slices and each piece was extracted with 0.5 ml of 50
mM Tris-HC1 buffer, pH 7.5, containing 30% glycerol at 4°C
overnight. The material extracted from each slice was assayed for kininase activity.
Preparation of Antibodies Anti-Endooligopeptidase A From
Rat Brain
Endooligopeptidase A was purified from rat brain minus
cerebellum (Wistar; male; 400 g) essentially as previously
described (13). The antibody against purified enzyme was
E N K E P H A L I N G E N E R A T I O N AND ENDOOLIGOPEPTIDASE A
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FIG. 1. DEAE-cellulose chromatography of the pH 5 supernatant
fraction of the heart homogenate. The sample (1.87 g of protein and
26.6 U in 870 ml) was equilibrated with 50 mM Tris HCI buffer pH
7.5 containing 30 mM NaC1 and applied to the column (2.5×70 cm)
equilibrated with the same buffer. The chromatography was carried
out at 5°C under a flow rate of 220 ml/hr; fractions of 9 ml were
collected. The elution of the proteins was achieved by stepwise
increase of the NaCI concentration in the buffer as indicated by the
arrows to 50, 70, 100 mM and 1.0 M, respectively. Kininase activity
was determined by bioassay with isolated guinea pig ileum ( 0 - - 0 )
and protein was monitored by UV absorbance at 280 nm (--). The
horizontal bars DE-I and DE-II indicate the pooled fractions under
each peak displaying kininase activity.
obtained by injecting subcutaneously 170 mU of enzyme
(specific activity 1600 mU/mg) into male New Zealand rabbits
using the scheme described in Camargo et al. (12) for the
bovine enzyme. The IgG fraction of the antiserum was
purified by DEAE-cellulose chromatography as the first
protein peak emerging from the column at pH 8.6 buffered
with 25 mM Tris-HC1 containing 35 mM NaCI (14). About 12
/xg of antibody thus purified is needed to inhibit 1.5 mU of
rabbit heart endooligopeptidase A.
SDS Gel Electrophoresis
Lyophilized salt-free protein samples were taken up in
100/zl of 48 mM Tris-HCl buffer, pH 6.7, containing 1% SDS
(w/v) and 6.5 mM of dithiothreitol, 0.5% bromophenol blue
and 10% glycerol (v/v). After heating for 3 min at 100°C the
samples were electrophoresed on a 7 to 15% linear gradient
acrylamide gel slab according to the method of Laemmli (26),
and stained as described above.
Immunoblotting
Immunoblotting was carried out essentially as described
by Towbin et al. (36). Briefly, samples dissolved in boiling
SDS-PAGE sample buffer containing 10% SDS and 10%
2-mercaptoethanol were electrophoresed in 6--16% polyacrylamide linear gradient microslab gels prepared according
to Matsudaira and Burgess (28). After electrophoresis
proteins were transferred to nitrocellulose sheets (Millipore,
0.45/xm pore size) for at least 2 hours at 200 mA. The trans-
FIG. 2. Gel filtration on Sephacryl S-200 of the fraction DE-I. The
pooled fractions under peak DE-I (Fig. 1) containing 11.6 U and 147
mg of protein were concentrated to 14 ml under reduced pressure at
5°C in an 8/32 Nojax Visking dialysis tube. This sample was applied
to a column (2.5× 110 cm) equilibrated and developed at 5°C with 50
mM Tris-HCl buffer, pH 7.5 containing 100 mM NaCI, at a flow rate
of 8.0 ml/hr. The horizontal bars S-I, S-II and S-Ill represent the
pooled fractions containing 18%, 12% and 65% of the total effluent
kininase activity, respectively. Kininase activity (O---O) and UV
absorbance at 280 mm (--) were monitored as described in Fig. 1.
ferred polypeptides and Mr markers were visualized by
staining the sheets with 0.1% (w/v) Ponceau-S in 10% acetic
acid. The sheets were subsequently soaked in blot buffer
(150 mM NaCI, 1 mM EDTA, 30 mM Tris/HCl pH 7.3, 0.25%
gelatin, 0.05% Tween 20 and 0.05% NAN3) for at least 30
minutes to block the remaining protein binding sites. After
incubation with the rabbit antiserum against rat brain
endooligopeptidase A diluted 1:50 and monoclonal antibody
against tubulin YOL 1/34 (25) ascitic fluid-diluted 1:200 in
blot buffer for 1 hr at room temperature, the sheets were
subjected to three washes of 15 minutes each in blot buffer
under constant motion. Bound immunoglobulins were visualized after incubation for 1 hr with the appropriate
antiimmunoglobulin antibody coupled to horseradish peroxidase following three 10 min washes in PBS and reaction with
diaminobenzidine (0.2 mg/ml) and H202 (5/zl of a 30% solution in 30 ml PBS).
Determination of Products Derived From Bradykinin,
Neurotensin and Enkephalin Containing Peptides
Two methods were applied to determine the products
formed when the peptides were incubated with heart peptidases. When bradykinin was used as substrate we applied
the method described by Carvalho and Camargo (13) which
consists of the application of the incubation mixture in an
automatic amino acid analyzer (1) programmed to measure
every arginine containing peptide derived from bradykinin.
The products derived from neurotensin and enkephalin containing peptides were determined by reversed-phase HPLC
on a Waters Associates system using a/~Bondaback C18 column (3.9×300 ram). The detailed experimental conditions
are described elsewhere (12).
CICILINI ET AL.
948
Enzyme Assays With Alpha-N-Benzoyl-Gly-Ala-Ala-Phe-pAmino-Benzoate
The enzyme assays for high Mr and low Mr
endooligopeptidase A were performed as described by Orlowski et al. (31) using alpha-N-benzoyl-Gly-Ala-Ala-Phep-aminobenzoate as substrate. In brief, the substrate (1 mM)
was incubated in a final volume of 200/xl of 200 mM TrisHCI, pH 7.0, at 25°C, containing 0.2 mM dithiothreitol and
0.2 mU of purified high Mr or low Mr endooligopeptidase A.
Incubations were performed at 37°C for 20 min and the reaction terminated by heat inactivation at 100°C for 2 min.
Samples were then cooled on ice prior to a further 2 hr incubation at 37°C initiated by addition of 50 tzl of 5 mM dithiothreitol containing 20/zg aminopeptidase M. The amount of
para-aminobenzoate released was determined by absorbance
at 550 nm following diazotization as described (31).
Protein and Peptide Concentration Measurements
Protein was determined by the method of Bensadoun and
Weinstein (4). The concentrations of low Mr endooligopeptidase A obtained from the acrylamide gel after electrophoresis and those of synthetic peptides were determined
by amino acid analysis. Aliquots from purified enzyme and
peptide solutions were lyophilized and subjected to acid
hydrolysis as described by Simpson et al. (33). Hydrolyzates
were analyzed using an automatic amino acid analyzer over a
Beckman W-3 ion-exchange column according to Alonzo
and Hirs (1). The amount of protein and peptides was calculated from the quantity of each amino acid residue determined by amino acid analysis.
RESULTS
Purification of Heart Endooligopeptidase A
The pH 5 supernatant fraction of rabbit extract containing
1,970 mg of protein (specific activity 6.3 m U/mg) was applied
on a DE-52 anion exchange column. The profile showed in
Fig. 1 indicates the presence of two major components of
bradykinin-inactivating activities eluted respectively at 50
and 70 mM NaC1. The solid bars at the bottom of Fig. 1
(DE-I and DE-II) indicate the pooled fractions. They correspond to 46% and 23% of the kininase activity introduced
into the DE-52 column, respectively. The increase in specific
activities was 5.7-fold for DE-I and 4.1-fold for DE-II. The
fragments released from bradykinin indicated that the DE-I
fraction contains endooligopeptidase A like activity since it
releases stoichiometric amounts of bradykininl_~ and
bradykinin6 9. The fragments released by the DE-II and III
fractions revealed the predominance of exopeptidases acting
on the carboxyl-terminus of bradykinin and was not further
examined. Gel filtration of DE-I fraction on Sephacryl S-200
resulted in two peaks of kininase activity (Fig. 2). The high
Mr peak was recovered in the void volume. The specific
activity of the pooled fractions of the high Mr enzyme (solid
bar, S-I in the bottom of Fig. 2) is 68.2 mU/mg protein. It
corresponds to 7.4% of the total kininase activity present in
the 25,000 × g × 60 min supernatant fraction of heart homogenate. The specific activity of the pooled fractions of the low
Mr enzyme (solid bar, S-III in the bottom of Fig. 2) is 148.6
mU/mg protein yielding 14.4% of the total activity present in
the 25,000xg supernatant fraction. The low Mr enzyme was
estimated to have an apparent Mr=71,000 by gel filtration on
Sephacryl S-200.
The analysis of the fragments generated when bradykinin
was incubated with the high Mr and low Mr enzymes
indicates that both released stoichiometric amounts
of bradykininl_,~ and bradykinin6 ~. The high Mr endooligopeptidase A enzyme loses 50% of kininase activity when
stored at -20°C in the presence of 30% glycerol within the
first 48 hours following gel filtration. Less than 10% of
enzyme activity is found after one week storage period at
-20°C in the presence or absence of 30% glycerol. On the
other hand, the low Mr endooligopeptidase A can be stored
for two months at -20°C in presence of 30% glycerol, without significant loss of activity.
The S-I, S-I1, S-III and DE-I fractions were further
purified by disc electrophoresis in polyacrylamide gels. Figure 3 shows the stained bands of proteins and the region
where kininase activities were detected. For the PAGE of
the high Mr enzyme the kininase activity is coincident with
the major protein band. No kininase activity was detected in
other segments of the gel. Attempts to extract the high Mr
enzyme from the gel resulted in very low recovery of enzyme
activity, usually less than 20%. Gel electrophoresis of the
low Mr enzyme (Fig. 3) resulted in a single region of kininase
activity where no visible band of protein could be observed.
In contrast with the high Mr enzyme, the recovery of low Mr
enzyme activity from the gel was about 80%. The yield of
purified low Mr endooligopeptidase A recovered from each
gel was 32 mU with specific activity of 3.8 U/mg. Figure 3
also shows the stained gels corresponding to fractions S-II
and DE-I. Both exhibited two regions containing kininase
activity which correspond to the activities found in the gels
of fractions S-I and S-III. The enzymes obtained by preparative gel electrophoresis were analyzed by SDS-PAGE. Figure 4 shows the stained gels of high Mr and low Mr preparations of endooligopeptidase A after SDS-PAGE. A band
corresponding to a polypeptide of Mr around 72,000 is observed in both high Mr and low Mr enzymes although several
other bands are detected in the high Mr form of the enzyme
by this procedure.
Immunochemical Relationships of Heart
Endooligopeptidase A, Brain Endooligopeptidase A and
Cytoskeleton Proteins
The immunoglobulin raised against rat brain endooligopeptidase A was tested against high Mr and low Mr
heart endooligopeptidase A by immunoblotting after SDSPAGE. Low Mr and high Mr heart enzymes showed one
major band of antigen with Mr=73,000 (Fig. 5, left) which is
similar to Mr of rabbit brain endooligopeptidase A (13). The
immunoblot of high Mr enzyme preparation exhibited two
other bands of Mr=58,000 and 15,000 which could correspond to degradation products of low Mr form of
endooligopeptidase A. It was also observed that high Mr
enzyme preparation contained substantial amounts of tubulin whereas this protein was not detected in the low Mr
enzyme (Fig. 5, right). In addition, the use of either a
monoclonal or an antiactin polyclonal antibody did not disclose the existence of any immunoreactive analogue of the
protein in both enzyme preparations (data not shown).
Hydrolysis of Neurotensin, Enkephalin Containing Peptides
and Alpha-N-Benzoyl-Gly-Ala-Ala-Phe-p-Aminobenzoate by
Heart High Mr and Low Mr Endooligopeptidase A
Due to the observation that brain endooligopeptidase A
ENKEPHALIN
GENERATION
AND ENDOOLIGOPEPTIDASE
DE-I.
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A
949
S-II
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FIG. 3. Polyacrylamide gel electrophoresis of high Mr and low Mr heart endooligopeptidase A. The gels were loaded with about 40 mU of enzyme, corresponding to
samples taken from fractions DE-I (Fig. 1), S-l, S-ll and S-Ill (Fig. 2), as indicated.
Electrophoresis was carried out at 5°C for 17 hr with 0.6 mA/tube on 6% acrylamide gels.
After the run the gels were stained with Coomassie blue and the position of the enzymatic
activity determined on gels ran in parallel, indicated by brackets in the figure. The
detailed procedures are described in the Experimental section.
950
CICIL1NI E T AL.
$td
High Mr LowHr
the Experimental Procedures section. When neurotensin,
BAM-12P and dynorphin B are incubated with either high Mr
or low Mr endooligopeptidase A the only products formed
are neurotensinl s and neurotensing_l.~ or the enkephalins
with the respective complements BAM-12P6_~z and dynorphin B6-~3. Figure 6 illustrates the HPLC profile of the
hydrolyzates of neurotensin, BAM-12P and dynorphin B by
high Mr enzyme. The peptides eluted in each peak were
collected and subjected to acid hydrolysis followed by amino
acid analysis (data not shown) and correspond in a integral
molar ratio to the following peptides: neurotensin~_8,
neurotensing_~:+, neurotensin, BAM-12PHe, BAM-12P and
enk(Met), dynorphin B6 1:~,dynorphin B and enk(Leu) (Fig.
6A, B and C).
Both high Mr and low Mr endooligopeptidase A can also
release enk(Leu) from neoendorphins (alpha and beta) and
from dynorphin A~_s (Fig. 7). In analogy with brain
endooligopeptidase A, the heart high Mr enzyme does not
hydrolyze large enkephalin containing peptides such as
BAM-22P and dynorphinl_~7. Similar results were obtained
for low Mr heart endooligopeptidase A (data not shown).
Table 1 shows the site of cleavage of the biologically active
polypeptides studied here. The peptide benzoyl-Gly-AlaAla-Phe-p-aminobenzoate was not hydrolyzed by either of
the high Mr and low Mr enzymes.
Effects o f Various Substances on High Mr and Low Mr
Endooligopeptidase A Activity
When the enzyme assays are carried out in the presence
of dithiothreitol and using dynorphin B as substrate, both
high Mr and low Mr are activated (Table 3). The enzymes
are fully inhibited by thiol reagents such as p-OHmercuribenzoate (1 mM) and 5,5'-dithiobis (2-nitrobenzoic) acid (3 raM) but not by 2 ~M leupeptin and by 10 mM
DFP. Less specific thiol reagents such as Zn ++ and Cu ++ at
0.5 mM, also display a very strong inhibitory effect.
Both low Mr and high Mr endooligopeptidase A are not
significantly affected by EDTA or by N-[l-(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, specific
inhibitor of the metalloendopeptidase EC.3.4.24.15 (14).
However, 11.8 /zg of anti-endooligopeptidase A immunoglobulins completely inhibit 1.3 mU of both low Mr and high
Mr enzymes.
DISCUSSION
FIG. 4. SDS-PAGE of purified high Mr and low Mr heart
endooligopeptidase A. The electrophoresis was performed on a
7-15% linear gradient slab gel and the protein bands detected by
Coomassie blue staining. Both high Mr and low Mr endooligopeptidase A samples were obtained by preparative gel elec-
trophoresis (Fig. 3). Mr markers ran in parallel correspond to the
following proteins: bovine serum albumin, ovalbumin, chymotrypsinogen and ribonuclease A. The detailed procedures are described
in the Experimental section.
cleaves the Arg8-Arg9 bond of NT (10), the MeP-Arg 6 bond of
BAM-12P (11) and the LeuS-Arg6 bond of dynorphin B (12)
we investigated the ability of both high Mr and low Mr
endooligopeptidase A from rabbit heart to hydrolyze these
neuropeptides. Peptide substrates were incubated with each
enzyme purified by gel electrophoresis and the reaction
medium analyzed by reversed phase HPLC as described in
In the present study, two endooligopeptidase A of different molecular weights were found in the cytosol of rabbit
heart. Both low Mr and high Mr enzymes exhibited the same
pattern of peptide bond cleavages of bradykinin, neurotensin
and on enkephalin containing peptides as compared to the
cysteinyl brain endooligopeptidase A. The presence of a high
Mr endooligopeptidase A in nervous tissue has also been
observed by Oliveira et al. (29).
Inclusion of bradykinin in the incubation mixture resulted
in a concentration dependent inhibition of enkephalin conversion by low Mr and high Mr heart endopeptidases (data
not presented) thus indicating that the same enzyme cleaves
both PheS-Ser6 bond of bradykinin and the MetS-Arg6 and
Leu~-Arg6 bonds of enkephalin containing peptides to release enkephalins. It is also worth noting that both low Mr
and high Mr heart endopeptidases cannot be considered
metallo- or serine-endopeptidases because they were not
significantly affected by EDTA or DFP. However, they were
E N K E P H A L I N G E N E R A T I O N AND ENDOOLIGOPEPTIDASE A
Low
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FIG. 5. Immunoblotting analyses of high Mr and low Mr rabbit heart endooligopeptidase A
using antibodies directed to rat brain endooligopeptidase A and to tubulin. Immunoblotting
was carried out as described in the Experimental Procedures section. Left Panel: Samples
of purified high Mr and low Mr enzymes as revealed by rabbit antibodies raised against
purified rat brain endooligopeptidase A. Right Panel: Samples of Trypanosoma cruzi (Tu)
whole cell extract and of both high Mr and low Mr enzyme preparations as revealed by
monoclonal (YOL 1/34) antitubulin antibody.
activated by dithiothreitol and fully inhibited by pOH-mercuribenzoate and by 5,5'-dithiobis (2-nitrobenzoic)
acid which characteristically affects cysteinyl-proteinases.
Endooligopeptidase A can also be distinguished from
cathepsins because the enzyme is inactive at acid pH and
does not hydrolyze denatured proteins (8,30). In addition,
leupeptin, which at 2/xm concentration completely inhibits
cathepsins [see (3)] showed no inhibitory effect towards
endooligopeptidase A. Similarly to the cysteinyl brain
endooligopeptidase A which cleaves only oligopeptides (9,
11, 12, 29) heart endopeptidases do not hydrolyze large
opioid peptides. The resistance of large peptides to
hydrolysis by heart enzymes seems to justify the general
name of endooligopeptidase applied to these endopeptidases.
The lack of susceptibility of large peptides and proteins to
hydrolysis by low Mr and high Mr endopeptidases clearly
discriminates between these enzymes and other multiple
forms of intracellular proteinases such as Ca++-activated
protease (41), Calpain (40) and multicatalytic proteinase (17).
Recently Chu and Orlowski (15) have described a soluble
metallo-endopeptidase EC.3.4.24.15 from rat brain with
specificity similar to endooligopeptidase A. However, heart
low Mr and high Mr endooligopeptidase A neither hydrolyze
the EC.3.4.24.15 synthetic substrate nor are inhibited by the
specific inhibitor of this enzyme (Table 2). Moreover, Chu
and Oriowski (15) did not detect the presence of
EC. 3.4.24.15 in the heart tissue where endooligopeptidase A
was found in large quantities (16). In addition, we have
demonstrated that brain endooligopeptidase A but not the
EC.3.4.24.15 was able to generate enkephalin from dynorphinl_s enk(Met)-Arg-Arg-Val-NH2 (metorphamide) (35).
Both enzymes were inhibited by the antiserum against rat
brain endooligopeptidase A thus indicating that they contain
at least some common epitopes.
We have previously demonstrated that brain endooligopeptidase A is a protein of a single peptide chain
of Mr=71,000 (13). A band of protein of similar Mr was also
obtained for heart low Mr endooligopeptidase A. However,
for high Mr enzyme the SDS gel electrophoresis showed
several other bands besides the Mr 72,000 band, thus indicating that high Mr enzyme preparation contains other proteins
which could be associated or contaminating the purified
enzyme. Nevertheless, the lack of polypeptides of higher Mr
suggests that high Mr form of endooligopeptidase A could
consist of an associative form of low Mr enzyme with other
cytosolic proteins.
C I C I L I N I ET A L
952
A
1!
!
OTT
B
OTT
OTT
1;
!
E
L
,4"
t,i
/
I
¢U
•
/
j f
/
n,,
I,-,.
I..lJ
o
z
I--
0
u')
m
4
w
fj
h
rr'
o
o
11
O_
5
10
15
5
10
TIME
15
5
10
15
(rain.)
FIG. 6. Hydrolysis of NT (Panel A), BAM-12P (Panel B) and dynorphin B (Panel C) by high Mr endooligopeptidase
A estimated by analysis over reversed phase HPLC. Peptides were separated by an initial isocratic elution in 0. I%
H:~PO4, pH 2.7, followed by a 15 rain linear gradient up to 35% acetonitrile in 0. I% H:~PO~, pH 2.7, at a flow rate of 2
ml/min, and monitored by UV absorbance at 214 nm. The chromatograms show the hydrolysis of NT (12 nmol),
BAM 12P (16 nmol) and dynorphin B (11 nmol) incubated in 1.0 ml of 50 nM Tris-HCl buffer, pH 7.5, containing 0.3
mM dithiothreitol at 37°C with 2.5 mU of high Mr endooligopeptidase A. The reaction was stopped by addition of 5
p.l of concentrated H:~PO~ to obtain 30 to 60% hydrolysis of substrate. The products separated by HPLC were
collected and subjected to amino acid analysis after acid hydrolysis. The identification of each peak is indicated in the
HPLC profiles. Dyn B, dynorphin B; DTT, dithiothreitol.
100-
"S
o
C
mr--_--,
50,
,
FIG. 7. Hydrolysis of enkephalin-containing peptides by purified
endooligopeptidase A. Ten to fifteen nmol of each of the following
synthetic substrates were incubated at 37°C with 1.3 mU of high Mr
enzyme in 1.0 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 0.3
mM dithiothreitol during 5, 10, 15, 30 and 45 min: neoendorphin
(alpha) (A), neoendorphin (beta) (&), dynorphin A1_8 ([]), dynorphin AI-1r (~), dynorphin B (©), BAM 12P ( I ) and BAM 22P (0).
The reaction was stopped by addition of 5/~1 of concentrated H 3 P O 4.
The products of the enzymatic reaction were analyzed over reversed
phase HPLC using an isocratic elution in 0.1% H 3 P O 4 , pH 2.7, containing 10% (v/v) acetonitrile followed by a linear gradient from 10%
to 50% acetonitrile in 0.1% H3PO4, pH 2.7 at flow rate of 2 ml/min.
Reaction products were monitored by absorbance at 214 rim.
m
i_
0
G.)
04
w
I
15
w
I
30
TIME (rain)
w
I
45
953
E N K E P H A L I N G E N E R A T I O N AND ENDOOLIGOPEPTIDASE A
TABLE 1
SITE OF CLEAVAGESOF PEPTIDESHYDROLYSEDBY HIGH Mr AND LOW Mr HEART
ENDOOLIGOPEPTIDASEA
T
Bradykinin
Neurotensin
Arg- Pro- Pro- Gly- Phe- Ser- Pro- Phe- Arg
<Glu-Leu-Tyr- Glu- Asn-Lys- Pro- Ar:Arg- Pro- Tyr- lie- Leu-
t
BAM-12P
Tyr- Gly- Gly- Phe- Met-Arg- Arg- Val- Gly- Arg- Pro- Glu
Dynorphin B
Tyr- Gly- Gly- Phe- Le~Arg- Arg- Gin- Phe- Lys- Val- Thr- Arg
Neoendorphin (alpha)
Tyr- Gly- Gly- Phe- Le~Arg- Lys- Tyr- Pro-
Neoendorphin (beta)
Tyr- Gly- Gly- Phe- Le~Arg- Lys- Tyr
Dynorphin A,
Tyr- Gly- Gly- Phe- Leu~-Arg- Arg- lie
The site of cleavage of peptide bond is indicated by an arrow.
TABLE 2
EFFECT OF COMPOUNDSON THE FORMATIONOF ENK(LEU) FROMDYNORPHIN B BY
HIGH Mr AND LOW Mr HEARTENDOOLIGOPEPTIDASEA
Substance
None
Dithiothreitol
5,5'-Dithiobis
(2-nitrobenzoic acid)
p-OH-mercuribenzoate
Leupeptin
DFP
EDTA
CF-A-A-F-pAB
ZnCI2
CuSO4
Anti-Brain-endooligopeptidase
A-IgG
Concentration
(raM)
Low Mr
Rel. Activity
(%)
High Mr
Rel. Activity
(%)
-0.2
0.4
0.8
1.5
3.0
0.5
1.0
0.002
10.0
1.0
2.0
0.3
0.5
0.5
11.8 tzg
100
110
140
130
25
0
15
0
100
84
100
85
90
0
0
0
100
120
130
110
35
0
20
0
100
78
80
65
100
0
0
0
The low Mr and high enzyme (1.3 mU) obtained by preparative gel electrophoresis was
preincubated with the indicated compounds in 0.05 M Tris HCI buffer, pH 7.5, for 15 min at
4°C. Dynorphin B (11 nmol) was added and incubated for l0 min at 37°C. The reaction was
stopped by addition of 5 /~l of concentrated HaPO4. Peptides were analysed by reversed
phase HPLC according to the method described in the legend to Fig. 7.
Somewhat indirect evidence that high Mr endooligopeptidase consists of an associative form of the low Mr
species of the enzyme was obtained by the immunoblot
analysis showed in Fig. 6. Both low Mr and high Mr enzyme
react with antienzyme antibody producing a band of around
73,000 daltons which is similar to the Mr of the rabbit brain
endooligopeptidase A 03).
An apparent discrepancy emerges from the analysis of
these results since the high Mr form of the enzyme was defined as the activity recovered in the exclusion volume of a
liquid chromatography sizing step and both SDS-PAGE and
the immunoblotting analysis indicated similar molecular
weights for low Mr and high Mr forms of endooligopeptidase
A. Since only major chemical modifications (such as carbohydrates) could yield differences in Mr on SDS-PAGE, we
believe that the behaviour of high Mr endooligopeptidase A
both in gel filtration and PAGE electrophoresis do reflect
associative characteristics of this species. The same immunoblotting experiments also show antigens of Mr 58,000
and 15,000 in the high Mr preparation which could corre-
954
C I C I L I N I ET A L .
spond to proteolytic fragments of the low Mr enzyme. This
presumed susceptibility to proteolysis could also explain the
low enzymatic stability of the high Mr enzyme described
above.
Other immunoblot studies also revealed the presence of
tubulin in the high Mr enzyme preparation. This finding
suggests that at least part of the endooligopeptidase A found
in the 25,000xg supernatant fraction could be associated
with cytoskeletal elements. It is a widespread observation to
find cytosolic protein in association with the cytoplasmic
matrix and the importance of such associations has been
extensively studied in the enzymes of the intermediary metabolism (5, 6, 21, 27).
One metabolic event which occurs during axonal transport is related to the processing of oxytocin and vasopressin
in the secretory neurons of hypothalamus (20). In this respect, the association of endooligopeptidase A with microtubules may represent an effective metabolic com-
partmentalization in order to achieve, for instance, localized
processing of opioid peptides. However, since this line of
experimental evidence cannot formally exclude artifacts
such as nonspecific associations or even nonphysiological
reassociation in vitro, the precise significance of the observations made here requires further investigations. Among
these are the direct demonstration of both physical and physiological associations between the enzyme tubulin and the
opioid peptides which could be achieved by careful coisolation studies and specific-labeling immunofluorescence
studies on isolated cardiac cells.
ACKNOWLEDGEMENTS
We thank Mr. Gerson B. Silva for technical help and Mrs. Yara
Corradini for skillful secretarial assistance. Supported by Fundaqao
de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP).
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