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Liver bradykinin-inactivating-endopeptidase is similar to the metalloendopeptidase (EC 3.4.24.15)
Immunopharmacology ELSEVIER
Irnmunopharmacology 32 (1996) 176-179
Liver bradykinin-inactivating-endopeptidase is similar to the metalloendopeptidase ( EC 3.4.24.15) Hercilia M. Molina a, Adriana K. Carmona b, Mafia Kouyoumdjian a, Durval R. Borges c,,, Luiz Juliano b b
Department of Biochemist~', Universidade Federal de S~o Paulo / EPM, S{to Paulo, Brazil Department of Btophystcs, Universidade Federal de S~to Paulo / EPM, S~to Paulo, Brazil c Department of Medicine, Unit,ersidade Federal de S~o Paulo/EPM, S~o Paulo, Brazil .
•
Abstract The bradykinin-inactivating-endopeptidase (BIE) removal from rat liver, by perfusing the organ with 0.05% Triton X-100, achieved its maximum at 10 min of perfusion and falls to 50% of the maximum in 30 rain, a pattern similar to AST removal. Using an internally quenched fluorescent BK analogue (Abz-RPPGFSPFRQ-EDDnp) we further characterized this enzyme: it is activated by low concentrations of 2-mercaptoethanol, inhibited by p-hydroxymercuribenzoate, o-phenanthroline and EDTA, and is resistant to enalapril, E-64 and PMSF. These results suggest that BIE is a metalloendopeptidase containing a thiol group important for its activity. BIE also hydrolyses the peptides Abz-GGFLRRVQ-EDDnp, AbzGPQGLAGQ-EDDnp, Abz-FRSVQ-EDDnp, and Abz-ARVRRANSFLQ-EDDnp. All these properties are very similar to those described or assayed by us for EC 3.4.24.15, isolated initially from rat testes and then from several organs of different animals. Both BIE and EC 3.4.24.15: hydrolyze the FSS 6 bond of the BK fluorescent substrate; are efficiently inhibited by Orlowski specific inhibitor (CFP-AAF-pAB, K i 4.4× l 0 - 7 M and 1.25 × 10 - 7 M, respectively); have the same electrophoretic mobility in SDS-PAGE (M r 78000); and are both recognized by three polyclonal antibodies raised against rat testes EC 3.4.24.15. In conclusion, BIE appears to be EC 3.4.24.15. Keywords: Bradykinin; Kininase: Liver; Metalloendopeptidase; Thimet; EC 3.4.24.15.
1. Introduction Bradykinin (BK) inactivation in the whole rat is a fast reaction, even when the pulmonary tissue is
Abbreviations: BK, bradykinin; BIE, bradykinin-inactivatingendopeptidase; Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine; ALT, alanine aminotransferase; AST, aspartate aminotransferase and LDH: lactate dehydrogenase; 2ME, 2-mercaptoethanol; E-64, (L-trans-epoxysuccinyl-L-Leu-cylamido(4-guanidino)butane * Corresponding author. Caixa Postal 20239, 04034-970 Sao Paulo, SP Brazil. E.mail: drborges.dmed@epm,br
bypassed (Prado et al., 1975). Following a single passage considerable amounts of BK are inactivated by the exsanguinated and perfused rat liver preparation (Borges et al., 1976) by a non-lysosomal thiol activated endopeptidase, that splits BK at the FSS 6 bond. This enzyme can he removed by perfusing the organ with Triton X-100 0.05%, (Borges et al., 1979). The hepatic bradykinin-inactivating endopeptidase characteristics are: pI 4.9, inhibition by 0.05 mM TPCK, 1 mM ortho-phenanthroline, 100 mM SQ 14225 and 1 mM DFP; partial inhibition by 1 m M EDTA, 1 m M EGTA; no inhibition by 0.5 mM
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved. SSDI 0 162-31 09(95 ) 0 0 0 8 6 - 0
H.M. Molina et al. / lmmunopharmacology 32 (1996) 176-179
T L C K and 5 mM sodium tetrathionate (Kouyoumdjian et al., 1984). These characteristics seemed similar to those of other oligoendopeptidases such as rat testis endopeptidase (EC 3.4.24.15) (Tisljar, 1993), brain endopeptidase A (Camargo et al., 1972), and a soluble metalloendopeptidase from rat brain (Orlowski et al., 1983). To further characterize BIE a fluorescent BK analogue (Abz-RPPGFSPFRQ-EDDnp) was used to compare it with the Thi-Met oligopeptidase (EC 3.4.24.15)
2. Material and methods
BIE was removed from perfused liver and purified from the perfusate as described by Kouyoumdjian et al. (1984). The following internally quenched fluorescent peptides were tested as substrates for BIE: Abz-GGFLRRV-EDDnp; Abz-GPQGLAGQEDDnp; A b z - F R S V Q - E D D n p ; A b z - A R V R RANSFLQ-EDDnp; Abz-RPPGFSPFRQ-EDDnp; Abz-GGFLRR-EDDnp; Abz-GGFLR-EDDnp; AbzLGPDRQ-EDDnp; Abz-GPLGPDRQ-EDDnp; AbzAGLAQ-EDDnp and Abz-TQLLVRQ-EDDnp. BIE activity was determined in a final volume of 2 ml of 50 mM Tris-HC1 (pH 8.0); after the addition of 50 /xl of fluorogenic peptide substrates (1 m g / m l ) the hydrolysis was monitored by measuring the fluorescence at AEM----420 nm and A z x = 3 2 0 nm in a Hitachi F-2000 spectrofluorimeter. Inhibitors used were pre-incubated at 37°C for 30 min with the enzyme before addition of Abz-RPPGFSPFRQ-EDDnp. The hydrolysis of Abz-RPPGFSPFR-EDDnp and Abz-GGFLRRV-EDDnp were followed by HPLC (Chagas et al., 1995). The scissile bond was determined by isolation of fragments by HPLC and the structure deduced from amino acid composition of each fragment. Samples of partially purified BIE were submitted to SDS/PAGE (10%) and then transferred to nitrocellulose (semi-dry electrophoretic transfer). The immunoblots were developed with three polyclonal antibodies against different forms of EC 3.4.24.15 as primary antibody and anti-rabbit immunoglobulin G-peroxidase as second antibody; both enzyme and antibodies kindly supplied by MJ Glucksman (Mount Sinai School of Medicine, New York, USA). The
177
immunoblots were then revealed with diaminobenzidine.
3. Results
The bradykinin-inactivating-endopeptidase (BIE) was removed from rat liver by perfusing the organ with 0.05% Triton X-100 in Krebs-Henseleit solution; the removal achieved its maximum at 10 min of perfusion and falls to 50% of the maximum in 30 min, a pattern similar to AST removal (Fig. 1); alkaline phosphatase and y-glutamyl transferase were not removed in the conditions used. We observed that BIE is: (a) activated by low concentrations (2.5-15 mM) of 2-mercaptoethanol (2ME) and (0.025-0.5 mM) of dithiothreitol; the effect is abolished at higher concentrations 50-300 and 1-3 mM, respectively (Fig. 2); (b) inhibited by 1 mM iodoacetamide, 0.1 mM mercuric acetate, 0.1 mM p-hydroxymercuribenzoate, 0.1 mM orthophenanthroline and 0.1 mM EDTA; (c) efficiently inhibited, as EC 3.4.24.15, by Orlowski's specific inhibitor (CPF-AAF-pAB): K i 4.4 × 10 7M (BIE) and 1.25 × 10 7 M (EC 3.4.24.15); and (d) not inhibited by 1 mM enalapril, 0.05 mM E-64 (L-
trans-epoxysuccinyl-L-Leu-cylamido(4-guanidino)butane, a thiol specific inhibitor) and 2 mM PMSF.
160 0.1~
140 120
•~
~"
1~04
40 ~ 2O h
I 20
t
I 40
P e ~
t
I
I
60
L 80
I
0 100
time (rain)
Fig. 1. R e m o v a l o f BIE a n d other e n z y m e s by the perfusion o f isolated rat liver with Triton X - 1 0 0 0.05%. n = times superior of a reference value. BIE, b r a d y k i n i n inactivating endopeptidase; A L T , alanine aminotransferase; AST, aspartate aminotransferase; and L D H , lactate d e h y d r o g e n a s e .
H.M. Molina et al./ lmmunopharmacology 32 (1996) 176-179
178 260 240
~
-!
220 200
160
t40 v
TT(reMit00)
120
:E too <"5
--o-- 2 r ~
F-
SO 60 40
0
~ilill 0
20
,Ilia[ Jllllii ] t,,i,t,l*,ll,i,] 40
60
80
l i t ~ it i I rid I t , Jl J ' l i l l ~
100 120 140 160 180 200 220 2AO 260 280 300
Concentration Fig. 2. Effect of thiol compounds on BIE activity.
Using various fluorescent substrates, we observed that BIE hydrolyses Abz-RPPGFSPFRQ-EDDnp, Abz-GGFLRRV-EDDnp, Abz-GPQGLAGQ-EDDnp, Abz-FRSVQ-EDDnp and Abz-ARVRRANSFLQ-EDDnp, but does not hydrolyze AbzGGFLRR-EDDnp, Abz-GGFLR-EDDnp, AbzLGPDRQ-EDDnp, Abz-GPLGPDRQ-EDDnp, AbzAGLAQ-EDDnp or Abz-TQLLVRQ-EDDnp. Cleavage positions of Abz-RPPGFSPFR-EDDnp and Abz-GGFLRRV-EDDnp by BIE were determined by isolation of fragments by HPLC. The structure deduced from amino acid composition of each fragment indicated that FSS 6 and LaR 5 bonds, respectively, were cleaved. BIE and EC 3.4.24.15 have the same electrophoretic mobility in S D S / P A G E (M r 78 000) and both enzymes are recognized by three polyclonal antibodies against different forms of the latter.
4. D i s c u s s i o n
A liver bradykinin-inactivating endopeptidase described by Borges et al. (1979) and Kouyoumdjian et al. (1984) has characteristics comparable to the oligoendopeptidase EC 3.4.24.15, a thiol-dependent metallo-endopeptidase widely distributed in animals (Tisljar, 1993). We now show that both BIE and the EC 3.4.24.15 enzyme (Lew et al., 1995) hydrolyze the same bond ( F s S 6) of the fluorescent substrate
BK. The inhibition pattern suggests that BIE is a metalloendopeptidase containing a thiol group important for its activity, similar to the one described for chicken liver Pz-peptidase (Barrett and Brown, 1990). BIE and 24.15 are both inhibited by Orlowski's specific inhibitor with similar Ki: 4.4 × 10 - 7 M and 1.25 × 10 - 7 M , respectively. BIE and EC 3.4.24.15 have the same electrophoretic mobility in SDS-PAGE (M r 78000) and are identical immunologically. BIE cleavage positions in some peptide substrates are similar to those of endopeptidase A (Juliano et al., 1990), as well as to those of a microsomal endopeptidase that processes vitamin K-dependent proteins (Kawabata and Davie, 1992). In conclusion, BIE seems to be EC 3.4.24.15.
References
Barrett AJ, Brown MA. Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase. Biochem J 1990; 271: 701-706. Borges DR, Prado JL, Limbos EA, Camargo ACM. Catabolism of vasoactive polypeptide by perfused rat liver. Naunyn-Schmied Arch Pharmacol 1976; 295: 33-40. Borges DR, Guimaraes JA, Limbos EA, Prado JL, Camargo ACM. Bradykinin inactivation by perfused rat liver: role of a thiol activated endopeptidase. Biochem Pharmacol 1979; 30: 1065-1069. Camargo ACM, Ramalho-Pinto FJ, Greene LJ. Brain peptidase: conversion and inactivation of kinin hormones. J Neurochem 1972; 19: 37-49. Chagas JR, Portaro FCV, Hirata IY, Almeida PC, Juliano MA, Juliano L, Prado ES. Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins. Biochem J 1995; 306: 63-69. Juliano L, Chagas JR, Hirata IY, Carmona E, Sucupira M, Oliveira ES, Oliveira EB, Camargo ACM. A selective assay for endooligopeptidase A based on the cleavage of fluorogenic snbstrate structurally related to enkephalin. Biocbem Biophys Res Commun 1990: 173: 647-652. Kawabata, Davie JBC. A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the Vitamin K-dependent proteins of plasma. J Biochem Chem 1992; 267: 10331-10336. Kouyoumdjian M, Borges DR, Prado JL. Kinin-inactivating endopeptidase from rat liver. Int J Biochem 1984; 16:733 739. Lew RA, Hey N J, Tetaz T J, Glucksman MJ, Roberts JL, Smith AI. Substrate specificity differences between recombinant rat testes endopeptidase EC 3.4.24.15 and the native brain enzyme. Biochem Biophys Res Commun 1995: 209: 788-795. Orlowski M, Michaud C, Chu TG. A soluble metalloendopeptidase from rat brain, purification of the enzyme and determina-
H.M. Molina et al. / Immunopharmacology 32 (1996) 176 179 tion of specificity with synthetic natural peptides. Eur J Biochem 1983; 135: 81-88. Prado JL, Limbos EA, Roblero J, Freitas JO, Prado ES, Paiva ACM. Recovery and conversion of kinins in exsanguinated rat preparation. Naunyn-Schmied. Arch Pharmacol 1975; 290: 191 21)5.
179
Tisljar U. Thimet oligopeptidase - a review of a thiol dependent metallo-endopeptidase also known as Pz-peptidase endopeptidase 24.15 and endo-oligopeptidase. Biol Chem Hoppe-Seyler, 1993: 374:91 11)0.
Irnmunopharmacology 32 (1996) 176-179
Liver bradykinin-inactivating-endopeptidase is similar to the metalloendopeptidase ( EC 3.4.24.15) Hercilia M. Molina a, Adriana K. Carmona b, Mafia Kouyoumdjian a, Durval R. Borges c,,, Luiz Juliano b b
Department of Biochemist~', Universidade Federal de S~o Paulo / EPM, S{to Paulo, Brazil Department of Btophystcs, Universidade Federal de S~to Paulo / EPM, S~to Paulo, Brazil c Department of Medicine, Unit,ersidade Federal de S~o Paulo/EPM, S~o Paulo, Brazil .
•
Abstract The bradykinin-inactivating-endopeptidase (BIE) removal from rat liver, by perfusing the organ with 0.05% Triton X-100, achieved its maximum at 10 min of perfusion and falls to 50% of the maximum in 30 rain, a pattern similar to AST removal. Using an internally quenched fluorescent BK analogue (Abz-RPPGFSPFRQ-EDDnp) we further characterized this enzyme: it is activated by low concentrations of 2-mercaptoethanol, inhibited by p-hydroxymercuribenzoate, o-phenanthroline and EDTA, and is resistant to enalapril, E-64 and PMSF. These results suggest that BIE is a metalloendopeptidase containing a thiol group important for its activity. BIE also hydrolyses the peptides Abz-GGFLRRVQ-EDDnp, AbzGPQGLAGQ-EDDnp, Abz-FRSVQ-EDDnp, and Abz-ARVRRANSFLQ-EDDnp. All these properties are very similar to those described or assayed by us for EC 3.4.24.15, isolated initially from rat testes and then from several organs of different animals. Both BIE and EC 3.4.24.15: hydrolyze the FSS 6 bond of the BK fluorescent substrate; are efficiently inhibited by Orlowski specific inhibitor (CFP-AAF-pAB, K i 4.4× l 0 - 7 M and 1.25 × 10 - 7 M, respectively); have the same electrophoretic mobility in SDS-PAGE (M r 78000); and are both recognized by three polyclonal antibodies raised against rat testes EC 3.4.24.15. In conclusion, BIE appears to be EC 3.4.24.15. Keywords: Bradykinin; Kininase: Liver; Metalloendopeptidase; Thimet; EC 3.4.24.15.
1. Introduction Bradykinin (BK) inactivation in the whole rat is a fast reaction, even when the pulmonary tissue is
Abbreviations: BK, bradykinin; BIE, bradykinin-inactivatingendopeptidase; Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine; ALT, alanine aminotransferase; AST, aspartate aminotransferase and LDH: lactate dehydrogenase; 2ME, 2-mercaptoethanol; E-64, (L-trans-epoxysuccinyl-L-Leu-cylamido(4-guanidino)butane * Corresponding author. Caixa Postal 20239, 04034-970 Sao Paulo, SP Brazil. E.mail: drborges.dmed@epm,br
bypassed (Prado et al., 1975). Following a single passage considerable amounts of BK are inactivated by the exsanguinated and perfused rat liver preparation (Borges et al., 1976) by a non-lysosomal thiol activated endopeptidase, that splits BK at the FSS 6 bond. This enzyme can he removed by perfusing the organ with Triton X-100 0.05%, (Borges et al., 1979). The hepatic bradykinin-inactivating endopeptidase characteristics are: pI 4.9, inhibition by 0.05 mM TPCK, 1 mM ortho-phenanthroline, 100 mM SQ 14225 and 1 mM DFP; partial inhibition by 1 m M EDTA, 1 m M EGTA; no inhibition by 0.5 mM
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved. SSDI 0 162-31 09(95 ) 0 0 0 8 6 - 0
H.M. Molina et al. / lmmunopharmacology 32 (1996) 176-179
T L C K and 5 mM sodium tetrathionate (Kouyoumdjian et al., 1984). These characteristics seemed similar to those of other oligoendopeptidases such as rat testis endopeptidase (EC 3.4.24.15) (Tisljar, 1993), brain endopeptidase A (Camargo et al., 1972), and a soluble metalloendopeptidase from rat brain (Orlowski et al., 1983). To further characterize BIE a fluorescent BK analogue (Abz-RPPGFSPFRQ-EDDnp) was used to compare it with the Thi-Met oligopeptidase (EC 3.4.24.15)
2. Material and methods
BIE was removed from perfused liver and purified from the perfusate as described by Kouyoumdjian et al. (1984). The following internally quenched fluorescent peptides were tested as substrates for BIE: Abz-GGFLRRV-EDDnp; Abz-GPQGLAGQEDDnp; A b z - F R S V Q - E D D n p ; A b z - A R V R RANSFLQ-EDDnp; Abz-RPPGFSPFRQ-EDDnp; Abz-GGFLRR-EDDnp; Abz-GGFLR-EDDnp; AbzLGPDRQ-EDDnp; Abz-GPLGPDRQ-EDDnp; AbzAGLAQ-EDDnp and Abz-TQLLVRQ-EDDnp. BIE activity was determined in a final volume of 2 ml of 50 mM Tris-HC1 (pH 8.0); after the addition of 50 /xl of fluorogenic peptide substrates (1 m g / m l ) the hydrolysis was monitored by measuring the fluorescence at AEM----420 nm and A z x = 3 2 0 nm in a Hitachi F-2000 spectrofluorimeter. Inhibitors used were pre-incubated at 37°C for 30 min with the enzyme before addition of Abz-RPPGFSPFRQ-EDDnp. The hydrolysis of Abz-RPPGFSPFR-EDDnp and Abz-GGFLRRV-EDDnp were followed by HPLC (Chagas et al., 1995). The scissile bond was determined by isolation of fragments by HPLC and the structure deduced from amino acid composition of each fragment. Samples of partially purified BIE were submitted to SDS/PAGE (10%) and then transferred to nitrocellulose (semi-dry electrophoretic transfer). The immunoblots were developed with three polyclonal antibodies against different forms of EC 3.4.24.15 as primary antibody and anti-rabbit immunoglobulin G-peroxidase as second antibody; both enzyme and antibodies kindly supplied by MJ Glucksman (Mount Sinai School of Medicine, New York, USA). The
177
immunoblots were then revealed with diaminobenzidine.
3. Results
The bradykinin-inactivating-endopeptidase (BIE) was removed from rat liver by perfusing the organ with 0.05% Triton X-100 in Krebs-Henseleit solution; the removal achieved its maximum at 10 min of perfusion and falls to 50% of the maximum in 30 min, a pattern similar to AST removal (Fig. 1); alkaline phosphatase and y-glutamyl transferase were not removed in the conditions used. We observed that BIE is: (a) activated by low concentrations (2.5-15 mM) of 2-mercaptoethanol (2ME) and (0.025-0.5 mM) of dithiothreitol; the effect is abolished at higher concentrations 50-300 and 1-3 mM, respectively (Fig. 2); (b) inhibited by 1 mM iodoacetamide, 0.1 mM mercuric acetate, 0.1 mM p-hydroxymercuribenzoate, 0.1 mM orthophenanthroline and 0.1 mM EDTA; (c) efficiently inhibited, as EC 3.4.24.15, by Orlowski's specific inhibitor (CPF-AAF-pAB): K i 4.4 × 10 7M (BIE) and 1.25 × 10 7 M (EC 3.4.24.15); and (d) not inhibited by 1 mM enalapril, 0.05 mM E-64 (L-
trans-epoxysuccinyl-L-Leu-cylamido(4-guanidino)butane, a thiol specific inhibitor) and 2 mM PMSF.
160 0.1~
140 120
•~
~"
1~04
40 ~ 2O h
I 20
t
I 40
P e ~
t
I
I
60
L 80
I
0 100
time (rain)
Fig. 1. R e m o v a l o f BIE a n d other e n z y m e s by the perfusion o f isolated rat liver with Triton X - 1 0 0 0.05%. n = times superior of a reference value. BIE, b r a d y k i n i n inactivating endopeptidase; A L T , alanine aminotransferase; AST, aspartate aminotransferase; and L D H , lactate d e h y d r o g e n a s e .
H.M. Molina et al./ lmmunopharmacology 32 (1996) 176-179
178 260 240
~
-!
220 200
160
t40 v
TT(reMit00)
120
:E too <"5
--o-- 2 r ~
F-
SO 60 40
0
~ilill 0
20
,Ilia[ Jllllii ] t,,i,t,l*,ll,i,] 40
60
80
l i t ~ it i I rid I t , Jl J ' l i l l ~
100 120 140 160 180 200 220 2AO 260 280 300
Concentration Fig. 2. Effect of thiol compounds on BIE activity.
Using various fluorescent substrates, we observed that BIE hydrolyses Abz-RPPGFSPFRQ-EDDnp, Abz-GGFLRRV-EDDnp, Abz-GPQGLAGQ-EDDnp, Abz-FRSVQ-EDDnp and Abz-ARVRRANSFLQ-EDDnp, but does not hydrolyze AbzGGFLRR-EDDnp, Abz-GGFLR-EDDnp, AbzLGPDRQ-EDDnp, Abz-GPLGPDRQ-EDDnp, AbzAGLAQ-EDDnp or Abz-TQLLVRQ-EDDnp. Cleavage positions of Abz-RPPGFSPFR-EDDnp and Abz-GGFLRRV-EDDnp by BIE were determined by isolation of fragments by HPLC. The structure deduced from amino acid composition of each fragment indicated that FSS 6 and LaR 5 bonds, respectively, were cleaved. BIE and EC 3.4.24.15 have the same electrophoretic mobility in S D S / P A G E (M r 78 000) and both enzymes are recognized by three polyclonal antibodies against different forms of the latter.
4. D i s c u s s i o n
A liver bradykinin-inactivating endopeptidase described by Borges et al. (1979) and Kouyoumdjian et al. (1984) has characteristics comparable to the oligoendopeptidase EC 3.4.24.15, a thiol-dependent metallo-endopeptidase widely distributed in animals (Tisljar, 1993). We now show that both BIE and the EC 3.4.24.15 enzyme (Lew et al., 1995) hydrolyze the same bond ( F s S 6) of the fluorescent substrate
BK. The inhibition pattern suggests that BIE is a metalloendopeptidase containing a thiol group important for its activity, similar to the one described for chicken liver Pz-peptidase (Barrett and Brown, 1990). BIE and 24.15 are both inhibited by Orlowski's specific inhibitor with similar Ki: 4.4 × 10 - 7 M and 1.25 × 10 - 7 M , respectively. BIE and EC 3.4.24.15 have the same electrophoretic mobility in SDS-PAGE (M r 78000) and are identical immunologically. BIE cleavage positions in some peptide substrates are similar to those of endopeptidase A (Juliano et al., 1990), as well as to those of a microsomal endopeptidase that processes vitamin K-dependent proteins (Kawabata and Davie, 1992). In conclusion, BIE seems to be EC 3.4.24.15.
References
Barrett AJ, Brown MA. Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase. Biochem J 1990; 271: 701-706. Borges DR, Prado JL, Limbos EA, Camargo ACM. Catabolism of vasoactive polypeptide by perfused rat liver. Naunyn-Schmied Arch Pharmacol 1976; 295: 33-40. Borges DR, Guimaraes JA, Limbos EA, Prado JL, Camargo ACM. Bradykinin inactivation by perfused rat liver: role of a thiol activated endopeptidase. Biochem Pharmacol 1979; 30: 1065-1069. Camargo ACM, Ramalho-Pinto FJ, Greene LJ. Brain peptidase: conversion and inactivation of kinin hormones. J Neurochem 1972; 19: 37-49. Chagas JR, Portaro FCV, Hirata IY, Almeida PC, Juliano MA, Juliano L, Prado ES. Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins. Biochem J 1995; 306: 63-69. Juliano L, Chagas JR, Hirata IY, Carmona E, Sucupira M, Oliveira ES, Oliveira EB, Camargo ACM. A selective assay for endooligopeptidase A based on the cleavage of fluorogenic snbstrate structurally related to enkephalin. Biocbem Biophys Res Commun 1990: 173: 647-652. Kawabata, Davie JBC. A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the Vitamin K-dependent proteins of plasma. J Biochem Chem 1992; 267: 10331-10336. Kouyoumdjian M, Borges DR, Prado JL. Kinin-inactivating endopeptidase from rat liver. Int J Biochem 1984; 16:733 739. Lew RA, Hey N J, Tetaz T J, Glucksman MJ, Roberts JL, Smith AI. Substrate specificity differences between recombinant rat testes endopeptidase EC 3.4.24.15 and the native brain enzyme. Biochem Biophys Res Commun 1995: 209: 788-795. Orlowski M, Michaud C, Chu TG. A soluble metalloendopeptidase from rat brain, purification of the enzyme and determina-
H.M. Molina et al. / Immunopharmacology 32 (1996) 176 179 tion of specificity with synthetic natural peptides. Eur J Biochem 1983; 135: 81-88. Prado JL, Limbos EA, Roblero J, Freitas JO, Prado ES, Paiva ACM. Recovery and conversion of kinins in exsanguinated rat preparation. Naunyn-Schmied. Arch Pharmacol 1975; 290: 191 21)5.
179
Tisljar U. Thimet oligopeptidase - a review of a thiol dependent metallo-endopeptidase also known as Pz-peptidase endopeptidase 24.15 and endo-oligopeptidase. Biol Chem Hoppe-Seyler, 1993: 374:91 11)0.