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Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidases in human adrenal tumors
Neuropeptides(1997)31 (6),585-588 © HarcourtBraceand CompanyLtd 1997
Hip.Arg-Phe-, Hip.Phe.Arg- and Hip.His.Leu.cleaving dipeptidyl carboxypeptidases in human adrenal tumors A. Z. R6nai*, J. Lengyelt, T. Nagyt, G. Orosz**, V. Adlesz*, K. Racz*, K. Magyar§ *Department of Pharmacology, *Isotope Laboratory, * 2nd Department of Internal Medicine, and §Department of Pharmacodynamics, Semmelweis University of Medicine, Budapest, **Research Group of Peptide Chemistry, L. E6tv6s University, Budapest, Hungary
Summary Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidase activities were measured in the supernatant ($2) and pellet (P2) fractions obtained by ultracentrifugation of human adrenal tumor preparations. Negligible enzyme activity was found in cortical tumor whereas highly significant activities were present in the P2 fractions of the two pheochromocytoma specimens. The hydrolysis rates, expressed in terms of the percent of added substrate were 58-66%/60 min for Hip-Phe-Arg, 55-58%/60 min for Hip-Arg-Phe and 19-30%/60 min for HipHis-Leu. The angiotensin-converting enzyme inhibitor, captopril, differentially inhibited the enzyme splitting Hip-His-Leu versus the one cleaving Hip-Arg-Phe; Hip-Phe-Arg is probably the substrate of both. It is concluded that the Hip-ArgPhe-cleaving enzyme in adrenomedullary tumor is probably identical to the purportedly novel dipeptidyl carboxypeptidase that we detected earlier in rabbit ear artery wall, which converts (MetS)-enkephalin-Arg6,Phe 7 to (MetS)-enkephalin.
INTRODUCTION In the late 1970s and early 1980s, while studying dipeptidyl carboxypeptidases involved in the biotransformation of (Met3)-enkephalin and related endogenous opioid peptides, several teams detected enzymes that shared some properties with but were clearly different from angiotensin-converting enzyme (ACE, EC 3.4.15.1) (e.g.1). Some of these enzymes such as the one designated as enkephalinase 2 have later been proved to be identical to already known peptidases (in this case, neutral endopeptidase, EC 3.4.24.11), whereas others are still not clearly identified. We detected first by pharmacological means a dipeptidyl carboxypeptidase activity in isolated rabbit ear artery which rapidly converted (MetS)-enkephalinReceived21 May 1997 Accepted 18 August 1997 Correspondence to: A. Z. RSnai, Department of Pharmacology, Semmelweis University of Medicine, PO Box 370, H-1445 Budapest, Hungary Tel: 36-1-210-4416; Fax: 36-1-210-4412; Email: ronand@ net.sote.hu
Arg6,Phe 7 to (MetS)-enkephalinff the conversion was also proved by biochemical method in membranes prepared from the arteries. The enzyme could be inhibited only by high (> 10-51V0 concentrations of the ACE inhibitor, captopril. Another ACE inhibitor, enalaprilate, was even less effective, whereas the 'enkephalinase' inhibitor, thiorphan, 4 had no effect et all? The most characteristic natural substrate was (MetS)-enkephalin-Arg6,PheT; bradykinin was also a substrate but, apparently, angiotensin I and (MetS)-enkephalin were not. ~ The C-terminally amidated synthetic derivative of (MetS)enkephalin-Arg6,Phe 7 was not hydrolyzed. The enzyme was non-endothelial; indirect evidence suggested a neural location. Following the logic of synthetic ACE substrate Hip-His-Leu, we synthetized Hip-Arg-Phe and Hip-Phe-Arg- (based on the C-terminal dipeptide of bradykinin). In solubilized membranes prepared from rabbit ear artery, we demonstrated the presence of a nonendothelial enzyme which exhibited a preference for Hip-Arg-Phe substrate compared with Hip-His-Leu; this enzyme was apparently absent from rabbit aorta. 6 Since
585
586
R6nai et al
adrenomedullary cells are related developmentally to sympathetic post-ganglionic neurons and the adrenal medulla is also rich in proenkephalin-derived opioid peptides, z we decided to use human pheochromocytoma specimens as enzyme source. By using the three synthetic substrates and the ACE inhibitor, captopril, we could differentiate an enzyme activity in particulate (P2) fractions of pheochromocytoma homogenates which had similar properties to the one detected in rabbit ear artery wall; this enzyme activity was not present in adrenocortical tumor preparation. MATERIALS AND METHODS Drugs
The substrates were synthetized by conventional solution-phase method at the Research Group of Peptide Chemistry, E6tv6s University, Budapest. All the products had at least 97% HPLC purity and the expected amino acid composition. Captopril was kindly provided by the EGIS Pharmaceuticals (Hungary). Nonidet P-40 was purchased from Boehringer Mannheim (Austria). All the other reagents/solvents were of analytical or HPLC grade and were obtained from Reanal (Hungary) or Aldrich Chemical Co. (Milwaukee, W1).
The enzyme reactions were run at 37°C in phosphate buffer (see above) in 250 ~1 volume. The reaction mixture contained 0.3 mg protein enzyme source (S2 or P2) and 0.5 mM substrate, and was incubated for 60 rain. The reaction was terminated by adding 200 ~tl methanol and the mixture was centrifuged at 12 000 gfor 15 min. 20 ~tl volumes of supernatants were used without further separation for determining hippuric acid (Hip) content. Hip content was determined in an isocratic HPLC(ISCO 2350, Lincoln, NE) UV detector- (ISCO V4, at 228 nm) system using a 250 x 4 man Nucleosfl 5 C18 column with a 20 x 4 mm precolumn (BST, Technical University, Budapest) and a 10 mM NaH2PO4: methanol = 5 : 4 mobile phase adjusted to pH 3.0 with phosphoric acid, at a flow rate of 1.0 ml/min. The retention time of authentic Hip was 4.Smin; for the quantiicafion a Shimadzu C-R6A integrator (Kyoto, Japan) was used. The regression of the calibration curve for Hip was constructed and the position of the regression line was checked each experimental day at at least 2 Hip concentrations. The quantity of tissue samples was enough to carry out 2 independent experiments of the same type from each specimen (i.e. nos 101,108 and 118); the incubation mixtures were run in duplicate. The hydrolysis rate was expressed uniformly as percent of substrate cleavage in 60 min.
Methods
The tumor specimens were provided by the Steriod Laboratory of the 2nd Department of Internal Medicine, Semmelweis University of Medicine. For the use of specimens, an internationally standardized and documented approval was obtained from the Ethical Committee of the University. All the experimental procedures with the coded samples (nos 101, 108 and 118) were carried out in a double-blind manner; the retrospective histological identification took place after the completion of biochemical evaluation. The specimens were stored at -79-80°C until use. The individual tissue samples (one at a time for a definite series) were powderized under liquid nitrogen, and were left for 30 min at room temperature. The powderized tissue was taken up in 5.0 ml of 100 m_lVl Tris buffer, pH Z8, containing 250 mlVl saccharose, 30 mM KC1 and 5.0 mlVI magnesium acetate, and centrifuged at 6 0 0 g for 5 min. The supematant (S~) was ultracentrifuged (UP-65, VEB MC3N, Leipzig, Germany) at 100 000 g for 60 rain, and the pellet (P2) was taken up in 0.5 ml of phosphate puffer, pH 8.3, containing 300 mlVI NaC1 and 0.5% (v/v) Nonidet P-40 as solubilizer. The protein concentration of supematant ($2) and pellet (P2) solution was determined by the Lowry method. 8 The appropriate dilutions were made and the enzyme sources were stored in divided form at -18-20°C until use. Neuropeptides (1997) 31(6), 585-588
RESULTS
In the pilot experiments, the substrate and enzyme concentration dependence as well as the time course of enzyme reaction were determined; the standard incubation conditions (0.5 mM substrate concentration, 0.3 mg protein/250 ~1 enzyme source, 60 mill incubation) were based on these results. Using adrenocortical tumor as enzyme source (no. 101), the substrate hydrolysis rate was ZOO~o~60min for HRF, 6.0%/60 rain for HFR and 0%/60 man for HHL in the P2 fraction, and 5.3%/60 min for HRF, 4.5%/60 min for HFR and 00/0/60 min for HHL in the S2 fraction (two independent series for each, samples run in duplicate). In the S2 fraction of pheochromocytoma specimens (nos 108 and 118), the percent hydrolysis in 60 mill was 5.20/0 for HRF, 4.1O/o for HFR and 0% for HHL. In the P~ fractions of samples 108 and 118 (in this order) the hydrolysis rates were 66.0 and 58.2%/60 min for HFR, 55.0 and 58.4% /60min for HRF and 30.0 and 18.9%/ 60 min for HHL. The ACE inhibitor, captopril, when used in the concentration range of 10-~-2 x 10-6 M inhibited the enzymes responsible for the cleavage of HHL and HRF In a differentiating fashion in concentrations below 10-7 M (Fig.). The inhibition of HHL hydrolysis was more effective than that of HRF. The differentiation was much sharper © Harcourt Brace and Company Ltd 1997
Adrenomedullary peptidases
N ° 118
N°108 100 ~
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100
.
!
g
587
so
80-
8o
60-
. . . . . . . . . . . . . . . . . . . . .
40
4O
'i
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/ lO-a
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10-7
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20
110_ 8
10-6
i
10-7
i
10-e
Concentration of captopril (M) Figure Inhibition of Hip-His-Leu-, Hip-Arg-Phe- and Hip-Phe-Arg-cleaving dipeptidyl carboxypeptidase activities by captopril in P2 fractions prepared from human pheochromocytoma specimens. Nos 108 and 118. Abscissa: captopril concentration. Ordinate: percent inhibition of hippuric acid (Hip) production. Code numbers indicate tissue samples obtained from different patients. *HHL (Hip-His-Leu), • HFR (Hip-Phe-Arg); + HRF (Hip-Arg-Phe). Points represent the mean of 2 independent experiments, each run in duplicate.
in sample no. 108 than in 118; the effectiveness of inhibition of HFR cleavage by captoprfl was between those of HHL and HRF substrates (Fig.). DISCUSSION
It was found that human adrenocortical tumor tissue had a rather insignificant Hip-His-Leu-, Hip-Arg-Phe- or HipPhe-Arg-cleaving dipeptidyl carboxypeptidase activity, whereas the Pa fractions prepared from pheochromocytoma specimens contained dipeptidyl carboxypeptidases (or endopeptidase(s) acting as such) cleaving all the three synthetic substrates. The captopril-inhibited Hip-HisLeu- hydrolysing activity could be attributed to the action of authentic ACE, confirming the earlier reports on this issue. 9 The identity of the other enzyme, which acted as a dipeptidyl carboxy-peptidase on its preferred synthetic substrate, Hip-Arg-Phe and was differentiated from ACE by captopfil, is less certain. It is an additional, though not quite helpful piece of information that HipPhe-Arg (the synthetic substrate containing bradykinin's C-terminal dipeptide), besides being a well-known substrate of ACE, 1° is apparently a substrate also of this enzyme. The enzyme is not likely to be identical either to the dipeptidyl carboxypeptidase referred to as PDP-3 TM or to neutral endopeptidase EC.3.4.24.1112 because (1) it does not hydrolyze Hip-His-Leu as does PDP-3 and (2) in contrast to neutral endopeptidase, though at considerably © Harcourt Brace and Company Ltd 1997
higher concentrations than against ACE, captopril is still an effective inhibitor. The Hip-Arg-Phe- hydrolyzing activity found in human pheochromocytoma tissue shares several characteristics with the enzyme detected previously in rabbit ear artery, 3,5,e though the body of evidence is still not enough to declare identity. Both enzymes are ready to remove -Arg-Phe or -Phe-Arg but not -His-Leu dipeptides from the C-terminus of respective substrates, and both could be inhibited by considerably higher captopril concentrations than those required against ACE. Now that a convenient source of the potentially novel peptidase appears to be available, we wish to characterize the enzyme more closely with other known inhibitors such as thiorphan and EDTA, as well as some novel di- and heptapeptide inhibitors which have been found to have good selectivity in the rabbit ear artery assay (R6nai, unpublished). Furthermore, keeping in mind that the C-terminal dipeptide of atrial natriuretic peptide (........Phe $ Arg-Tyr) is also a potential cleaving site, we wish to include the synthetic substrate Hip-Arg-Tyr in further experiments. ACKNOWLEDGEMENTS
This work was supported by the National Fund for Scientific Research (OTKA) grate,-01 77 49. For the technical preparation of the manuscript, thanks are due to Ms Christine Barna. Neuropeptides (1997) 31(6), 585-588
588
R6naietal
REFERENCES 1. Cushman D W, Gordon E M, Wang F L, Cheung H S, Tung R, Delaney N G. Purification and characterization of enkephalinase, angiotensin converting enzyme, and a third peptidyldipeptidase from rat brain. Life Sci 1983; 33 (suppl I) 25-28. 2. Swerts J P, Perdtisot R, Malfroy B, Schwartz J C. Is 'enkephalinase' identical with 'angiotensin-converting enzymes'? Eur J Pharmacol 1979; 53: 209-210. 3. R6nai A Z, Serf6z6 P, HArsing L G Jr, Vizi E S. The conversion of met-enkephalin-Arg6,Phe 7 to met-enkephalin in rabbit ear artery. Life Sci 1983; 33 (suppl I): 101-104. 4. Llorens C, Gacel G, Swerts J P et al. Rational design of enkephalinase inhibitors: substrate specificity of enkephalinase studied from inhibitor potency of various peptides. Biochem Biophys Res Commun 1980; 96: 1710-1716. 5. R6nai A Z, Feh~r E, Boty~inszki J, Hepp J, Magyar A Medzihradszky K. Substrate- and inhibitor-specificity of a nonendothelial enzyme which forms [MetS]-enkephalin from [Met~]-enkephalin-Arg6,Phe 7 in isolated rabbit ear artery: pharmacological characterization. Neuropeptides 1995; 28: 137-145.
Neuropeptides (1997) 31(6), 585-588
6. Medzihradszky-Schweiger H, R6nai A Z, K&ay E, Medzihradszky K. Synthetic substrate and inhibitor profile of a dipeptidyl-carboxypeptidase in rabbit ear artery. In: Maia HLS ed. Peptides 1994; Leiden: ESCOM 1995: 893-894. 7. Kilpatrick D L, Jones B N, Lewis R Vet al. An 18,200-dalton adrenal protein that contains four [Met]-enkephalin sequences. Proc Natl Acad Sci USA 1982; 79: 3057-3061. 8. Lowry O H, Rosebrough N J, Farr A L, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1952; 193: 265-275. 9. Farsang C, Alf61di A. Increased ACE activity in pheochromocytoma. VIIIth Int. Congr. Endocrinology, Kyoto, 1988: 38Z abstr 14-21-031. 10. Erd6s E G, Slddgel R A. The unusual specificity and distribution of human angiotensin-I-converting enzyme. Hypertension 1986; 8 (suppl I): 134-13Z 11. Lanzillo J J, Dasarathy Y, Stevens J, Fanburg B L. Conversion of angiotensin-1 to angiotensin-2 by a latent endothelial cell peptidyl dipeptidase that is not angiotensin-converting enzyme. Biochem Biophys Res Commun 1986; 134: 770-776. 12. Erd6s E G, Skidgel R A. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEBJ 1989; 3: 145-151.
© Harcourt Brace and Company Ltd 1997
Hip.Arg-Phe-, Hip.Phe.Arg- and Hip.His.Leu.cleaving dipeptidyl carboxypeptidases in human adrenal tumors A. Z. R6nai*, J. Lengyelt, T. Nagyt, G. Orosz**, V. Adlesz*, K. Racz*, K. Magyar§ *Department of Pharmacology, *Isotope Laboratory, * 2nd Department of Internal Medicine, and §Department of Pharmacodynamics, Semmelweis University of Medicine, Budapest, **Research Group of Peptide Chemistry, L. E6tv6s University, Budapest, Hungary
Summary Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidase activities were measured in the supernatant ($2) and pellet (P2) fractions obtained by ultracentrifugation of human adrenal tumor preparations. Negligible enzyme activity was found in cortical tumor whereas highly significant activities were present in the P2 fractions of the two pheochromocytoma specimens. The hydrolysis rates, expressed in terms of the percent of added substrate were 58-66%/60 min for Hip-Phe-Arg, 55-58%/60 min for Hip-Arg-Phe and 19-30%/60 min for HipHis-Leu. The angiotensin-converting enzyme inhibitor, captopril, differentially inhibited the enzyme splitting Hip-His-Leu versus the one cleaving Hip-Arg-Phe; Hip-Phe-Arg is probably the substrate of both. It is concluded that the Hip-ArgPhe-cleaving enzyme in adrenomedullary tumor is probably identical to the purportedly novel dipeptidyl carboxypeptidase that we detected earlier in rabbit ear artery wall, which converts (MetS)-enkephalin-Arg6,Phe 7 to (MetS)-enkephalin.
INTRODUCTION In the late 1970s and early 1980s, while studying dipeptidyl carboxypeptidases involved in the biotransformation of (Met3)-enkephalin and related endogenous opioid peptides, several teams detected enzymes that shared some properties with but were clearly different from angiotensin-converting enzyme (ACE, EC 3.4.15.1) (e.g.1). Some of these enzymes such as the one designated as enkephalinase 2 have later been proved to be identical to already known peptidases (in this case, neutral endopeptidase, EC 3.4.24.11), whereas others are still not clearly identified. We detected first by pharmacological means a dipeptidyl carboxypeptidase activity in isolated rabbit ear artery which rapidly converted (MetS)-enkephalinReceived21 May 1997 Accepted 18 August 1997 Correspondence to: A. Z. RSnai, Department of Pharmacology, Semmelweis University of Medicine, PO Box 370, H-1445 Budapest, Hungary Tel: 36-1-210-4416; Fax: 36-1-210-4412; Email: ronand@ net.sote.hu
Arg6,Phe 7 to (MetS)-enkephalinff the conversion was also proved by biochemical method in membranes prepared from the arteries. The enzyme could be inhibited only by high (> 10-51V0 concentrations of the ACE inhibitor, captopril. Another ACE inhibitor, enalaprilate, was even less effective, whereas the 'enkephalinase' inhibitor, thiorphan, 4 had no effect et all? The most characteristic natural substrate was (MetS)-enkephalin-Arg6,PheT; bradykinin was also a substrate but, apparently, angiotensin I and (MetS)-enkephalin were not. ~ The C-terminally amidated synthetic derivative of (MetS)enkephalin-Arg6,Phe 7 was not hydrolyzed. The enzyme was non-endothelial; indirect evidence suggested a neural location. Following the logic of synthetic ACE substrate Hip-His-Leu, we synthetized Hip-Arg-Phe and Hip-Phe-Arg- (based on the C-terminal dipeptide of bradykinin). In solubilized membranes prepared from rabbit ear artery, we demonstrated the presence of a nonendothelial enzyme which exhibited a preference for Hip-Arg-Phe substrate compared with Hip-His-Leu; this enzyme was apparently absent from rabbit aorta. 6 Since
585
586
R6nai et al
adrenomedullary cells are related developmentally to sympathetic post-ganglionic neurons and the adrenal medulla is also rich in proenkephalin-derived opioid peptides, z we decided to use human pheochromocytoma specimens as enzyme source. By using the three synthetic substrates and the ACE inhibitor, captopril, we could differentiate an enzyme activity in particulate (P2) fractions of pheochromocytoma homogenates which had similar properties to the one detected in rabbit ear artery wall; this enzyme activity was not present in adrenocortical tumor preparation. MATERIALS AND METHODS Drugs
The substrates were synthetized by conventional solution-phase method at the Research Group of Peptide Chemistry, E6tv6s University, Budapest. All the products had at least 97% HPLC purity and the expected amino acid composition. Captopril was kindly provided by the EGIS Pharmaceuticals (Hungary). Nonidet P-40 was purchased from Boehringer Mannheim (Austria). All the other reagents/solvents were of analytical or HPLC grade and were obtained from Reanal (Hungary) or Aldrich Chemical Co. (Milwaukee, W1).
The enzyme reactions were run at 37°C in phosphate buffer (see above) in 250 ~1 volume. The reaction mixture contained 0.3 mg protein enzyme source (S2 or P2) and 0.5 mM substrate, and was incubated for 60 rain. The reaction was terminated by adding 200 ~tl methanol and the mixture was centrifuged at 12 000 gfor 15 min. 20 ~tl volumes of supernatants were used without further separation for determining hippuric acid (Hip) content. Hip content was determined in an isocratic HPLC(ISCO 2350, Lincoln, NE) UV detector- (ISCO V4, at 228 nm) system using a 250 x 4 man Nucleosfl 5 C18 column with a 20 x 4 mm precolumn (BST, Technical University, Budapest) and a 10 mM NaH2PO4: methanol = 5 : 4 mobile phase adjusted to pH 3.0 with phosphoric acid, at a flow rate of 1.0 ml/min. The retention time of authentic Hip was 4.Smin; for the quantiicafion a Shimadzu C-R6A integrator (Kyoto, Japan) was used. The regression of the calibration curve for Hip was constructed and the position of the regression line was checked each experimental day at at least 2 Hip concentrations. The quantity of tissue samples was enough to carry out 2 independent experiments of the same type from each specimen (i.e. nos 101,108 and 118); the incubation mixtures were run in duplicate. The hydrolysis rate was expressed uniformly as percent of substrate cleavage in 60 min.
Methods
The tumor specimens were provided by the Steriod Laboratory of the 2nd Department of Internal Medicine, Semmelweis University of Medicine. For the use of specimens, an internationally standardized and documented approval was obtained from the Ethical Committee of the University. All the experimental procedures with the coded samples (nos 101, 108 and 118) were carried out in a double-blind manner; the retrospective histological identification took place after the completion of biochemical evaluation. The specimens were stored at -79-80°C until use. The individual tissue samples (one at a time for a definite series) were powderized under liquid nitrogen, and were left for 30 min at room temperature. The powderized tissue was taken up in 5.0 ml of 100 m_lVl Tris buffer, pH Z8, containing 250 mlVl saccharose, 30 mM KC1 and 5.0 mlVI magnesium acetate, and centrifuged at 6 0 0 g for 5 min. The supematant (S~) was ultracentrifuged (UP-65, VEB MC3N, Leipzig, Germany) at 100 000 g for 60 rain, and the pellet (P2) was taken up in 0.5 ml of phosphate puffer, pH 8.3, containing 300 mlVI NaC1 and 0.5% (v/v) Nonidet P-40 as solubilizer. The protein concentration of supematant ($2) and pellet (P2) solution was determined by the Lowry method. 8 The appropriate dilutions were made and the enzyme sources were stored in divided form at -18-20°C until use. Neuropeptides (1997) 31(6), 585-588
RESULTS
In the pilot experiments, the substrate and enzyme concentration dependence as well as the time course of enzyme reaction were determined; the standard incubation conditions (0.5 mM substrate concentration, 0.3 mg protein/250 ~1 enzyme source, 60 mill incubation) were based on these results. Using adrenocortical tumor as enzyme source (no. 101), the substrate hydrolysis rate was ZOO~o~60min for HRF, 6.0%/60 rain for HFR and 0%/60 man for HHL in the P2 fraction, and 5.3%/60 min for HRF, 4.5%/60 min for HFR and 00/0/60 min for HHL in the S2 fraction (two independent series for each, samples run in duplicate). In the S2 fraction of pheochromocytoma specimens (nos 108 and 118), the percent hydrolysis in 60 mill was 5.20/0 for HRF, 4.1O/o for HFR and 0% for HHL. In the P~ fractions of samples 108 and 118 (in this order) the hydrolysis rates were 66.0 and 58.2%/60 min for HFR, 55.0 and 58.4% /60min for HRF and 30.0 and 18.9%/ 60 min for HHL. The ACE inhibitor, captopril, when used in the concentration range of 10-~-2 x 10-6 M inhibited the enzymes responsible for the cleavage of HHL and HRF In a differentiating fashion in concentrations below 10-7 M (Fig.). The inhibition of HHL hydrolysis was more effective than that of HRF. The differentiation was much sharper © Harcourt Brace and Company Ltd 1997
Adrenomedullary peptidases
N ° 118
N°108 100 ~
.
.
.
.
.
.
.
.
.
.
.
.
.
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100
.
!
g
587
so
80-
8o
60-
. . . . . . . . . . . . . . . . . . . . .
40
4O
'i
.
/ lO-a
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.
r
10-7
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.
.
.
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.
.
.
.
.
i
20
110_ 8
10-6
i
10-7
i
10-e
Concentration of captopril (M) Figure Inhibition of Hip-His-Leu-, Hip-Arg-Phe- and Hip-Phe-Arg-cleaving dipeptidyl carboxypeptidase activities by captopril in P2 fractions prepared from human pheochromocytoma specimens. Nos 108 and 118. Abscissa: captopril concentration. Ordinate: percent inhibition of hippuric acid (Hip) production. Code numbers indicate tissue samples obtained from different patients. *HHL (Hip-His-Leu), • HFR (Hip-Phe-Arg); + HRF (Hip-Arg-Phe). Points represent the mean of 2 independent experiments, each run in duplicate.
in sample no. 108 than in 118; the effectiveness of inhibition of HFR cleavage by captoprfl was between those of HHL and HRF substrates (Fig.). DISCUSSION
It was found that human adrenocortical tumor tissue had a rather insignificant Hip-His-Leu-, Hip-Arg-Phe- or HipPhe-Arg-cleaving dipeptidyl carboxypeptidase activity, whereas the Pa fractions prepared from pheochromocytoma specimens contained dipeptidyl carboxypeptidases (or endopeptidase(s) acting as such) cleaving all the three synthetic substrates. The captopril-inhibited Hip-HisLeu- hydrolysing activity could be attributed to the action of authentic ACE, confirming the earlier reports on this issue. 9 The identity of the other enzyme, which acted as a dipeptidyl carboxy-peptidase on its preferred synthetic substrate, Hip-Arg-Phe and was differentiated from ACE by captopfil, is less certain. It is an additional, though not quite helpful piece of information that HipPhe-Arg (the synthetic substrate containing bradykinin's C-terminal dipeptide), besides being a well-known substrate of ACE, 1° is apparently a substrate also of this enzyme. The enzyme is not likely to be identical either to the dipeptidyl carboxypeptidase referred to as PDP-3 TM or to neutral endopeptidase EC.3.4.24.1112 because (1) it does not hydrolyze Hip-His-Leu as does PDP-3 and (2) in contrast to neutral endopeptidase, though at considerably © Harcourt Brace and Company Ltd 1997
higher concentrations than against ACE, captopril is still an effective inhibitor. The Hip-Arg-Phe- hydrolyzing activity found in human pheochromocytoma tissue shares several characteristics with the enzyme detected previously in rabbit ear artery, 3,5,e though the body of evidence is still not enough to declare identity. Both enzymes are ready to remove -Arg-Phe or -Phe-Arg but not -His-Leu dipeptides from the C-terminus of respective substrates, and both could be inhibited by considerably higher captopril concentrations than those required against ACE. Now that a convenient source of the potentially novel peptidase appears to be available, we wish to characterize the enzyme more closely with other known inhibitors such as thiorphan and EDTA, as well as some novel di- and heptapeptide inhibitors which have been found to have good selectivity in the rabbit ear artery assay (R6nai, unpublished). Furthermore, keeping in mind that the C-terminal dipeptide of atrial natriuretic peptide (........Phe $ Arg-Tyr) is also a potential cleaving site, we wish to include the synthetic substrate Hip-Arg-Tyr in further experiments. ACKNOWLEDGEMENTS
This work was supported by the National Fund for Scientific Research (OTKA) grate,-01 77 49. For the technical preparation of the manuscript, thanks are due to Ms Christine Barna. Neuropeptides (1997) 31(6), 585-588
588
R6naietal
REFERENCES 1. Cushman D W, Gordon E M, Wang F L, Cheung H S, Tung R, Delaney N G. Purification and characterization of enkephalinase, angiotensin converting enzyme, and a third peptidyldipeptidase from rat brain. Life Sci 1983; 33 (suppl I) 25-28. 2. Swerts J P, Perdtisot R, Malfroy B, Schwartz J C. Is 'enkephalinase' identical with 'angiotensin-converting enzymes'? Eur J Pharmacol 1979; 53: 209-210. 3. R6nai A Z, Serf6z6 P, HArsing L G Jr, Vizi E S. The conversion of met-enkephalin-Arg6,Phe 7 to met-enkephalin in rabbit ear artery. Life Sci 1983; 33 (suppl I): 101-104. 4. Llorens C, Gacel G, Swerts J P et al. Rational design of enkephalinase inhibitors: substrate specificity of enkephalinase studied from inhibitor potency of various peptides. Biochem Biophys Res Commun 1980; 96: 1710-1716. 5. R6nai A Z, Feh~r E, Boty~inszki J, Hepp J, Magyar A Medzihradszky K. Substrate- and inhibitor-specificity of a nonendothelial enzyme which forms [MetS]-enkephalin from [Met~]-enkephalin-Arg6,Phe 7 in isolated rabbit ear artery: pharmacological characterization. Neuropeptides 1995; 28: 137-145.
Neuropeptides (1997) 31(6), 585-588
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