Peptide inhibitors of converting enzyme

Pergamon Press

Life Sciences, Vol . 21, pp . 1179-1186 Printed in the U.S .A .

PEPTIDE INHIBITORS OF CONVERTING ENZYME* Annette Fitz, Stephen Wyatt, David Boaz**, and Barbara Fox Department of Medicine, University of Iowa and Veterans Administration Hospital, Iowa City, Iowa 52240, U .S .A . (Received in final form September 12, 1977)

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the ren n-angiotensin system, and by angiotensin II and its analogues . Des-Asp l II (angiotensin III) 0 .1 mM and tridecapeptide renin substrate 0 .1 mM are both effective inhibitors human lung, plasma and porcine plasma converting enzymes . Des-Asp -Arg2 angiotensin II also was an effective inhibitor of plasma enzymes . Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate . The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system .

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Plasma angiotensin I converting enzyme (peptidyl dipeptidase [E .C . 3 .4 .14 .1 ;7]) and a bradykininase enzyme (kininase II) obtained from various tissues are thought to be identical (1-4) . Antibody raised against purified porcine renal angiotensin I converting enzyme cross-reacts with the lung and plasma converting enzymes from the hog (5) . Despite the presumed identity of these enzymatic activities, and the contrasting physiologic activities of the renin-angiotensin system and the kallikrein-kinin system, relatively little is known about the effects of other compounds of the renin-angiotensin system on the action of angiotensin converting enzyme or the enzyme's bradykininase activity . Angiotensin I has been shown to inhibit hydrolysis of bradykinin by converting enzyme (6) . Histidylleucine, the product of hydrolysis of angiotensin I by converting enzyme, and angiotensin III (des-Asp angiotensin II) are inhibitory to converting enzyme activity, as is angiotensin II (1,7) . Bradykinin and Phe-Arg, a product of bradykinin hydrolysis, are also inhibitory to the action of the enzyme in converting angiotensin I to angiotensin II (1,8,9) . Because of the clear, close relationship of the enzymatic activities which in physiologic systems might be considered to be complementary, we wished to study the effects of these and other peptides and peptide fragments of the angiotensin system on converting enzyme and bradykininase activities .

*Supported by National Institutes of Health Grant #GMB-2-ROI-HL-12684, and by the Veterans Administration Hospital, Research and Education Division . **Present address : 3-M Industrial Tapes Division, 3-M Center, St . Paul, Minn . 55101 1179

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Methods All common chemicals used were of reagent grade . Ultrapure enzyme grade ammonium sulfate, sucrose, apoferritin (equine), glutamic dehydrogenase, gamma globulin (human), ceruloplasmin (human), bradykinin, angiotensin II, angiotensin II hepta-, hexa- and pentapeptides, angiotensin I, renin tetradecapeptide substrate and tridecapeptide substrate were obtained from Schwarz/Mann, Orangeburg, New York . 1-sar-8-Ile angiotensin II was obtained from Beckman Instruments Co . (Spinco Division), Palo Alto, California . 1-sar-8-ala angiotensin II (3207-DCF-27) was obtained by courtesy of Norwich Pharmaceutical, Norwich, New York . Sepharose 6-B and Blue Dextran 2,000 as well as Sephadex columns, flow adaptors and eluent reservoirs, were obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey . Angiotensin I (5-ile) [leu 3,4,5-3H(N)], specific activity 250 mCi/mM, or 81 Ci/mM, angiotensin II (5-L-ile) [ile- C(U)], specific activity 237 mCi/mM, and bradykinin (bradykinin triacetate) [2-prolyl3,4-3H(N)] ; specific activity 43 .6 Ci/mM, were obtained from New England Nuclear, Boston, Mass . Thyroglobulin (porcine) was purchased from Sigma Chemical Company, St . Louis, Missouri . Thin-layer media type ITLC-SG was obtained from Gelman Instrument Company, Ann Arbor, Michigan . Scintillation rade dimethyl [2,2'-p-phenylenebis (4-methyl-5-phenyl)-oxazole] POPOP and PPO ?2,5-diphenyloxazole) were obtained from Fisher Scientific Company, Pittsburgh, Pennsylvania . Converting Enz Xmes : Four forms of angiotensin I converting enzyme, two typical angiotensin I converting enzymes obtained from human and porcine plasma and two large molecular weight "atypical" converting enzyme preparations obtained from human lung, were utilized in the study . The extraction and purification of angiotensin I [Phe8-His9] hydrolase (APHH) or "atypical" converting enzyme has been previously reported in detail (10,11) . Human cadaveric lung obtained at autopsy was cleared of blood with isotonic saline, homogenized in 0 .25 M sucrose, and fractionally precipitated three times with ammonium sulfate between 25% and 80% of saturation . Further purification was accomplished by ultracentrifugation at 75,000 x g followed by application to a Enzyme fractions were eluted with 0 .02 Sephadex 6-B column (2 .5 x 100 cm) . molar phosphate-A315% sodium chloride buffer, pH 6 .9 . Two active APHH fractions were obtained from the columns . The molecular weights of - these fractions were approximately 450,000 from the G-200 column and 600,000 and 450,000 from the Sepharose 6-B column . Human plasma converting enzyme was obtained by three ammonium sulfate precipitations followed by batch filtration on CM Sephadex C-50 utilizing sodium acetate buffer at pH 5 .5 . The enzyme was then further purified by chromatography on a DEAE-Sephadex A50 column, 0 .1 M Tris-HCL buffer pH 6 .0, utilizing Tris buffer and 0 .5 M NaCl to develop the gradient, followed by gel filtration on G-200 Sephadex, utilizing phosphate buffer pH Partially 6 .9 . Molecular weight of this material was approximately 206,000 . purified porcine converting enzyme was obtained from Miles Laboratory and was further purified utilizing G-200 Sephadex gel filtration with a phosphatechloride buffer (12) . Molecular weight of this material was approximately 260,000 . Enzyme Incubation : An enzyme sample, 0 .05 ml, 10-80 ug protein, was incubated or 0 minutes at 37° C with either 0 .025 ml (9 .9 x 10 - 1 nM), 250 mCi/mM [3H] angiotensin I or 0 .25 ml (5 .1 x 10 - 3 nM), 43 .6 Ci/mM [ 3 H] bradykinin, in phosphate buffer, pH 6 .9, containing 0 .0315% sodium chloride and 0 .00625% Inhibitor human albumin . Peptide inhibitors were made up in similar buffer . studies were performed by preincubating 0 .05 ml of enzyme with 0 .075 ml of the desired inhibitor for 30 minutes at 37° C . The isotopes were then added and the incubations continued for an additional 60 minutes . Samples were then frozen and assayed by electrophoresis or thin-layer chromatography . Products of the incubations of the enzymes with various substrates were separated by

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high-voltage electrophoresis at pH 3 .5 in a Savant Model FP-22A electroLyophilized incubates together with standards of angiotensin I, phorator . angiotensin II, and bradykinin as well as his-leu and leucine markers were spotted on Whatman #3MM chromatography paper,,23 x 43 cm . In the bradykinin assays cold bradykinin was spotted, dried and then the incubate was superimposed by a similar spotting technique and dried . Electrophoresis was performed in pyridine acetate buffer, pH 3 .5, for one hour 45 minutes at 3,000 volts x 90 milliamps . The position of the standard peptides and fragments was detected by spraying the paper with Sigma stock ninhydrin #3 . After drying at room temperature the paper was cut into strips 4 x 27 cm and scanned for radioactivity utilizing a Packard Chromatogram scanner . The strips were cut into pieces 0 .5 x 4 cm and each piece was subsequently assayed for radioactivity by scintillation spectrophotometry . The radio-purity of the substrates was also determined by this technique . The presence of a free 3 H leu peak, easily separated from other components AI, All and dipeptide, would indicate the presence of an angiotensinase or dipeptidase if present . Enzyme preparations used in these experiments had no detectable angiotensinase or dipeptidase activity . Liquid scintillation counting was performed in a Searle Model 6872 Isocap/300 temperature controlled liquid scintillation counter, utilizing glass vials with 10 ml solution containing 4 g PPO, 50 mg dimethyl POPOP, and made up to one liter with toluene . Converting enzyme activity was expressed as percent determined by comparing counts in the hisleu peak with total counts on the electrophoresis strip . The activity of converting enzyme was confirmed by bioassay on a superfused rat colon (13) . Bradykininase activity is likewise assessed by comparing the counts in the peak containing labeled bradykinin fragments with the counts in the standard [ H] labeled intact bradykinin peak, and total counts (13) . Results Two atypical converting enzyme fractions were obtained from human lung tissue - one of an approximate molecular weight of 600,000, the second of 450,000 . The effects of product inhibition of the converting enzymes utili zing angiotensin II and hisleu as well as angiotensin II derivatives and fragments and renin tetradecapeptide and tridecapeptide are shown in Table I . Angiotensin II 1 mM inhibited the "converting enzyme" for both the 600,000 and 450,000 molecular weight lung enzymes by more than 75% . Addition of the dipeptide hisleu in the incubation mixture, however, had little effect on the lung converting enzyme activity . The substituted angiotensin II inhibitors, 1-sar-8-ile angiotensin II and 1-sar-8-ala angiotensin II, exhibited different inhibitory effects with 8-ile derivative inhibiting 30% of the action of converting enzyme on angiotensin I and the 8-ala derivative having little effec~ on this reaction . In contract, the angiotensin II hetapeptide 1 mM (des-Asp angiotensin II or angiotensin III) inhibited converting enzyme, by ore than 75% and by 20% at 0 .01 mM . The angiotensin II hexapeptide (des-Asp -Arg angiotensin II) 1 mM inhibited more than 65% of converting enzyme activity and the pentapeptide (des-Asp l-Arg 2 -Val3 angiotensin II) 1 mM inhibited 50% . The bradykinin potentiating factor nonapeptide at 0 .1 mM concentrations inhibited 19% of converting enzyme activity in both preparations ; the BPF pentapeptide 0 .1 mM inhibited 15% of the 600,000 MW material as well . Renin tetradecapeptide substrate 0 .1 mM which was shown in an earlier study to be a substrate for both the 450,000 and 600,000 molecular weight fractions, inhibited the conversion of angiotensin I to angiotensin II by both the 600,000 and 450,000 Mw enzymes . Likewise, tridecapeptide renin substrate 1 mM inhibited by 60% the reaction of the 600,000 molecular weight enzyme fraction . Porcine and human plasma converting enzymes were inhibited by approximately 30% by angiotensin II . The porcine enzyme was slightly inhibited by

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the dipeptide hisleu, but the human plasma enzyme was unaffected (Table II) . Substantial inhibition was found with the addition of the angiotensin II heptapeptide and the hexapeptide derivatives . 1-sar-8-ile angiotensin II also inhibited porcine and human plasma converting enzyme activity . TDP, tridgcageptide renin substrate, bradykinin, the BPF nova- and pentapeptides and 3Phe, 8 Tyr angiotensin II were inhibitory to porcine converting enzyme . The inhibitory effect of the tridecapeptide renin substrate was particularly striking for the human plasma converting enzyme where 73% of the conversion of angiotensin I to II was inhibited . Table I Peptides as Inhibitors of "Atypical" Human Lung Converting Enzyme % His-Leu Released from Substrate 450,000 Mw

600,000 MR Peptide 5-Ile-angiotensin II 1 mM 5-Ile-angiotensin II 0 .1 mM 5-Ile-angiotensin II 0 .01 mM His-Leu Dipeptide 1 mM 1-Sar-8-Ile Angiotensin II 1 mM 1-Sar-8 Alanine Angiotensin II 1 mM Angiotensin II (2-8) Heptapeptide 1 mm Angiotensin II (2-8) Heptapeptide 0 .01 MM Angiotensin II (3-8) Hexapeptide 1 mM Angiotensin II (3-8) Hexapeptide 0 .01 mM Angiotensin II (4-8) Pentapeptide 1 mM Angiotensin II (4-8) Pentapeptide 0 .01 MM Tetradecapeptide Renin Substrate 1 mM Tetradecapeptide Renin Substrate 0 .1 mM Tridecapeptide Renin Substrate 0 .1 mM Tridecapeptide Renin Substrate 0 .01 mM BPF Nonapeptide 0 .1 mM BPF Pentapeptide 0 .1 mM

Control

Inhibitor

Control

Inhibitor

59 .3 61 .5 54 .1 74 .6

12 .3 40 .8 52 .6 76 .0

30 .6 29 .4 30 .6 26 .8

6 .8 16 .5 30 .0 25 .1

62 .5

44 .1

30 .1

10 .3

74 .6

72 .9

26 .8

21 .4

59 .3

7 .0

30 .6

7 .4

54 .1

39 .1

30 .6

20 .2

59 .3

19 .0

30 .6

9 .6

54 .1

52 .9

30 .6

28 .4

59 .3

29 .2

30 .6

12 .8

54 .1

53 .7

30 .6

30 .5

83 .8

55 .8

53 .5

5 .9

55 .8

45 .6

53 .5

22 .4

45 .7

14 .7

----

----

45 .7 59 .3 42 .2

44 .4 48 .7 34 .0

---30 .6 ----

---24 .9 ----

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Table II Peptides as Inhibitors of Human and Porcine Plasma Converting Enzyme % His-Leu Released from Substrate Human Plasma CE Inhibitor 5-Ile-angiotensin II 0 .1 mM Angiotensin II (2-8) Heptapeptide 0 .1 mM Angiotensin II (3-8) Hexapeptide 0 .1 mM Angiotensin II (4-8) Pentapeptide 0 .1 mM His-Leu Dipeptide 0 .1 mm 1-Sar-8 Ile Angiotensin II 0 .1 mM 3-Phe-8-Tyr Angiotensin II 0 .1 mM Tetradecapeptide Renin Substrate 0 .1 mM Tridecapeptide Renin Substrate 0 .1 mM Bradykinin 0 .1 mM BPF Nonapeptide 0 .1 mM BPF Pentapeptide 0 .1 mM

Porcine Plasma CE

Control

Inhibitor

Control

Inhibitor

55 .7

36 .9

45 .4

31 .3

55 .7

27 .9

45 .4

13 .5

55 .7

35 .4

45 .4

26 .7

55 .7 55 .7

50 .9 53 .7

45 .4 45 .4

44 .2 33 .8

55 .7

29 .5

45 .4

21 .9

----

----

44 .0

22 .1

55 .7

26 .4

45 .4

17 .8

55 .7 55 .7 55 .7 55 .7

15 .0 3 .2 7 .6 6 .4

45 .4 45 .4 45 .4 45 .4

14 .9 2 .9 2 .4 4 .4

Table III Effect of Peptides as Inhibitors of Bradykininase Activity % Bradykinin Destroyed Human Plasma CE Inhibitor Angiotensin I 0 .1 mM Angiotensin II 0 .1 mM His-Leu Dipeptide 0 .1 MM 1-Sar-8 Ile Angiotensin II 0 .1 mM Angiotensin II (2-8) Heptapeptide 0 .1 mM Angiotensin II (3-8) Hexapeptide 0 .1 mM Angiotensin II (4-8) Pentapeptide 0 .1 mM Tetradecapeptide Renin Substrate 0 .1 mM Tridecapeptide Renin Substrate 0 .1 mM BPF Nonapeptide 0 .1 mM BPF Pentapeptide 0 .1 mM

Porcine Plasma CE

Control

Inhibitor

Control

Inhibitor

51 .3 48 .8 48 .8

16 .8 30 .1 46 .4

35 .5 38 .6 38 .6

20 .6 23 .8 37 .4

51 .3

32 .8

35 .5

30 .0

51 .3

20 .7

35 .5

16 .7

51 .3

32 .6

35 .5

20 .2

51 .3

40 .6

35 .5

27 .0

48 .8

16 .2

38 .6

13 .7

48 .8 48 .8 48 .8

14 .0 5 .0 5 .7

38 .6 38 .6 38 .6

10 .6 6 .6 11 .0

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The inhibition of kininase activity of the human plasma converting enzyme and porcine plasma converting enzyme fractions was also studied . Angiotensin II was an effective inhibitor of the bradykininase activity, inhibiting 38% of the plasma converting enzyme activity from both human and porcine sources whereas the dipeptide hisleu 0 .1 mM also was ineffective as an inhibitor (Table III) . Renin tetradecapeptide substrate and tridecapeptide substrate were both very effective inhibitors, inhibiting over 65% and 70% of bradykininase respectively, as were the bradykinin potentiating factors nona- and pentapeptides . Likewise, angiotensin I was an effective inhibitor of bradykininase activity as was the 1-sar-8-ileu angiotensin II, and the hepta- and hexapeptides . The pentapeptide angiotensin II inhibited only 20% of the bradykininase activity . Discussion The renin-angiotensin system and the kallikrein-kinin system are both There is indirect evidence that the function of of physiologic significance . the renin-angiotensin-aldosterone system may influence urinary kallikrein (14) and strong evidence that one of the enzymes which inactivates bradykinin Bradykinin has (kininase II) is identical to angiotensin converting enzyme . vasodepressor, vasodilator and natriuretic functions, while the renin-angiotensin system is vasopressor . Therefore, in physiologic systems the bradykininase and angiotensin converting functions of the enzyme would be complementary . Angiotensin convertin enzyme has also been reportyd to form angiotensin III (angiotensin II (2-8~ heptapeptide) from (des-Asp ) angiotensin I, therefore, an alternative pathway for the formation of angiotensin III exists, at least in animals (7,15) . This pathway might assume physiologic significance since angiotensin III also serves as an effective inhibitor to converting enzyme derived from human lung, human plasma and porcine plasma . We have previously shown that "atypical" human lung converting enzyme (APHH) forms angiotensin II directly from tetradecapeptide renin substrate (16) . In view of the evident close relationship of the renin-angiotensin system, and the kallikrein-kinin system, and the evident pivotal role of angiotensin converting enzyme (kininase II) we wished to determine whether other portions of the renin-angiotensin system would be inhibitory to both the kininase and converting enzyme actions of the enzyme . As expected, both angiotensin II and angiotensin III were effective in inhibiting both the conversion of angiotensin I to angiotensin II and the destruction of bradykinin . Of some interest is the finding that the substituted angiotensin II analogues and angiotensin II heptapeptide fragment are equal to the parent angiotensin II as inhibitors of both converting enzyme and Oparil et al . had previously shown that the and bradykininase activities . amino acid found at the 8 position in the angiotensin structure was an important determinate of the action of converting enzyme (17) . The data presented in this paper, in conjunction with that of other investigators, suggests that the P!-terminal is also important in the action of converting enzyme . In addition, both tetra- and tridecapeptide renin substrates are effective inhibitors of both the kininase and converting enzyme activities . Although neither the tetradecapeptide nor the tridecapeptide substrate are known to occur naturally, they were the first obtained by tryptic digestion of natural renin substrate and there has been speculation that these compounds If this were the case, then the could be formed naturally in tissues (18) . inhibitory effect of these compounds could also be of major physiologic significance . Chiu et al . ( ;) have suggested that converting enzyme has a greater affinity for des-Asp angiotensin I than for angiotensin I itself, the data presented here showing that both the angiotensin II heptapeptide and the

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tridecapeptide renin substrate are converting enzyme inhibitors tends to indirectly support the observations of Chiu et al . References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .

H .Y .T . YANG, E .G . ERDOS, and Y . LEVIN, J . Pharm . Exp . Ther . 11 7 291-300 (1971) . E .G . ERDOS, Circ . Res . 36 247-255 (1975) . T . NAKAJIMA, G . OSHIMA, H.S .J . YEH,, R . IGIC, and E .G . ERDOS, Biochim . e t Bio h s . Acta 315 430-438 (1973) . M . DAS and R .LSUFFER, . J . Biol . Chem . 250 6762-6768 (1975) . G . OSHIMA, A . GECSE, and E .G . ERD S, Biochim . e t Biophys . Acta 350 26-37 (1974) . V .A . ALABASTER and Y .S . BAKHLE, Br . J . Pharm . 47 799-807 (1973) . A .T . CHIU, J .W . RYAN, J .M . STEWART, and F .E . RORER, Biochem . J . 155 189-192 (1976) . R . IGIC, E .G . ERDOS, H .S .J . YEH, K . SORRELLS, and T . NAKAJIMA, Circ . Res . Suppl . II Vol 31 51-61 (1972) . G .E . SANDER, D .W . WEST, and C .G . HUGGINS, Biochim . Biophys . Acta 242 662-667 (1971) . A . FITZ and M . OVERTURF, Hypertension 1972 , p . 507-511, Springer-Verlag, Berlin Heidelberg (1972) . D . BOAZ, S . WYATT, and A . FITZ, Fed . Proc . _33 1234 (1974) . Med . 103 295-299 (1956) . L .T . SKEGGS, J .R . KAHN, and N .P . -S HUF(W1(~~J Ex M . OVERTURF, S . WYATT, D . BOAZ, and A . FITZ, Life Sci . 161669-1682 (1975) . N .A .A . MACFARLANE, A . ADETUYIBI, and I .H . MILLS, J . Endocrin . 61 lxxii (1974) . A . LARNER, E .D . VAUGHAN, JR ., B .S . TSAI, and M .J . PEACH, Proc . Soc . Exp . Biol . Med . 15 2 631-634 (1976) . D . BOAZ, S . WYATT, and A . FITZ, Biochem . Biophys . Res . Commun . 6 3 490-495 (1975) . S . OPARIL, T . KOERNER, and J .K .O'DONOGHUE, Circ . Res . 34 19-26 (1974) . R . BOUCHER, I . ASEELIN, J . GENEST, Circ . Res . Suppl 1 Vol 34 203-209 (1974) .