Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.

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Metabolism of enkephalin in stomach wall of rats

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1990.258.1.g143 ◽

1990 ◽

Vol 258 (1) ◽

pp. G143-G151

Author(s):

N. W. Bunnett ◽

J. H. Walsh ◽

H. T. Debas

Keyword(s):

Angiotensin Converting Enzyme ◽

Stomach Wall ◽

Substantial Contribution ◽

Converting Enzyme ◽

In Vitro Degradation ◽

Bond Degradation ◽

Hydrolysis Of ◽

Specific Inhibitors

Peptidases degrade neuropeptides and thereby limit the duration and extent of their influence. This investigation examined the importance of peptidases in the degradation of the neuropeptide enkephalin in the stomach wall of the rat. Metabolism of [Leu5]- and [D-Ala2][Leu5]enkephalin by gastric membranes was examined in vitro. Degradation of [Tyr1-3H][Leu5]enkephalin was studied in the gastric submucosa of anesthetized and conscious rats in vivo by using a catheter to deliver peptide to tissues and implanted dialysis fibers to collect the metabolites. Specific inhibitors were used to assess the contribution of particular enzymes. [Leu5]- and [Tyr1-3H][Leu5]enkephalin were metabolized by membranes and in the stomach wall by hydrolysis of the Tyr1-Gly2 bond. Degradation was inhibited by the aminopeptidase inhibitor amastatin (10(-5) M in vitro, 10 nmol in vivo). Inhibitors of endopeptidase-24.11 (phosphoramidon) and angiotensin-converting enzyme (captopril) did not inhibit degradation. Metabolism of the aminopeptidase-resistant analogue [D-Ala2][Leu5]enkephalin by membranes was unaffected by amastatin and weakly inhibited by phosphoramidon affected by amastatin and weakly inhibited by phosphoramidon and captopril. A carboxypeptidase removed the COOH-terminal leucine residue and made a substantial contribution to degradation of both peptides by gastric membranes.

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Kinin B1 receptor antagonists with multi-enzymatic resistance properties

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y02-053 ◽

2002 ◽

Vol 80 (4) ◽

pp. 287-292 ◽

Cited By ~ 31

Author(s):

Witold Neugebauer ◽

Paul A Blais ◽

Stephanie Hallé ◽

Catherine Filteau ◽

Domenico Regoli ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Human Platelets ◽

Converting Enzyme ◽

Receptor Antagonists ◽

Rabbit Lung ◽

Duration Of Action ◽

Prolonged Duration ◽

Bradykinin Antagonists

The kinin B1 receptor has been implicated in a variety of pathological states; therefore, potent, selective, and specific antagonists with prolonged duration of action in vivo are needed. Using R-715 (AcLys[D-β-Nal7,Ile8] desArg9BK) as a template, new peptides containing α-MePhe in position 5, Oic in position 2, and AcOrn instead of AcLys at the N-terminal were prepared and tested for their antagonist potency, their selectivity, and their specificity for the kinin B1 receptor. In vitro metabolic stabilities toward aminopeptidase M (from human plasma), aminopeptidase P (from human platelets), and angiotensin-converting enzyme (purified from rabbit lung) were also investigated. The results of this study indicate that the three modifications applied separately are as well tolerated as they are when present conjointly in the template R-715. Indeed, pA2 values of R-715 (ranging from 8.40 to 8.5) do not differ significantly from the analogues R-954 and R-955 (both ranging from 8.4 to 8.6) when measured at kinin B1 receptors from rabbit aortas and human umbilical veins. Moreover, the chemical modifications utilized in the peptides R-954 and R-955 have provided resistance against aminopeptidases M and P, as well as the angiotensin-converting enzyme, unlike the early (e.g., Lys[Leu8]desArg9BK) and more recent (e.g., R-715, B-9858) generations of B1 receptor antagonists. Ongoing in vivo assays will validate the assumption that the analogues R-954 and R-955 have a prolonged duration of action.Key words: bradykinin, antagonists, B1 receptors, peptidases, metabolism.

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Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine

Biochimica et Biophysica Acta (BBA) - Enzymology ◽

10.1016/0005-2744(76)90045-0 ◽

1976 ◽

Vol 429 (1) ◽

pp. 220-228 ◽

Cited By ~ 41

Author(s):

Frederic E. Dorer ◽

Joseph R. Kahn ◽

Kenneth E. Lentz ◽

Melvin Levine ◽

Leonard T. Skeggs

Keyword(s):

Angiotensin Converting Enzyme ◽

Enzyme Hydrolysis ◽

Kinetic Properties ◽

Converting Enzyme ◽

Hydrolysis Of

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Endopeptidase 3.4.24.11 converts N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme

Biochemical Journal ◽

10.1042/bj2940681 ◽

1993 ◽

Vol 294 (3) ◽

pp. 681-684 ◽

Cited By ~ 19

Author(s):

C H Williams ◽

T Yamamoto ◽

D M Walsh ◽

D Allsop

Keyword(s):

Angiotensin Converting Enzyme ◽

Potent Inhibitor ◽

Neutral Endopeptidase ◽

Converting Enzyme ◽

Inhibitory Effects ◽

Rabbit Lung ◽

Pig Kidney ◽

Tissue Extracts ◽

Ic50 Value

It was reported recently that N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide (CPP-A-A-F-pAB), an inhibitor of endopeptidase 3.4.24.15 (E-24.15), also inhibits angiotensin-converting enzyme (ACE) from rabbit lung. We have found that this compound is without effect on ACE purified from pig kidney, at a concentration some 1000-fold greater than the Ki reported for inhibition of the enzyme from lung. However, preincubation of CPP-A-A-F-pAB with neutral endopeptidase 3.4.24.11 (E-24.11) does result in potent inhibitory effects on ACE. We have shown this to be due to formation of a fragment, CPP-A-A, the structure of which is closely related to ACE inhibitors such as enalaprilat. CPP-A-A was found to be a potent inhibitor of pig ACE. Under the conditions used it had an IC50 value of 1.6 x 10(-8) M, compared with the value obtained for captopril of 7.5 x 10(-10) M. These results have important implications for studies of E-24.15 when using CPP-A-A-F-pAB in vivo or in crude tissue extracts where E-24.11 might also be present.

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Transport, In Vivo Antihypertensive Effect, and Pharmacokinetics of an Angiotensin-Converting Enzyme (ACE) Inhibitory Peptide LVLPGE

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.0c07048 ◽

2021 ◽

Vol 69 (7) ◽

pp. 2149-2156

Author(s):

Jingyan Pei ◽

Ying Hua ◽

Tingyi Zhou ◽

Xinchang Gao ◽

Yali Dang ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Antihypertensive Effect ◽

Converting Enzyme ◽

Inhibitory Peptide ◽

Ace Inhibitory Peptide ◽

Ace Inhibitory

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Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00001.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. L162-L169 ◽

Cited By ~ 40

Author(s):

Kai Nowak ◽

Sandra Weih ◽

Roman Metzger ◽

Ronald F. Albrecht ◽

Stefan Post ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Weight Ratio ◽

Dry Weight ◽

Serum Levels ◽

Converting Enzyme ◽

Pulmonary Endothelium ◽

Preferential Expression

Limitation of reactive oxygen species-mediated ischemia-reperfusion (I/R) injury of the lung by vascular immunotargeting of antioxidative enzymes has the potential to become a promising modality for extension of the viability of banked transplantation tissue. The preferential expression of angiotensin-converting enzyme (ACE) in pulmonary capillaries makes it an ideal target for therapy directed toward the pulmonary endothelium. Conjugates of ACE monoclonal antibody (MAb) 9B9 with catalase (9B9-CAT) have been evaluated in vivo for limitation of lung I/R injury in rats. Ischemia of the right lung was induced for 60 min followed by 120 min of reperfusion. Sham-operated animals (sham, n = 6) were compared with ischemia-reperfused untreated animals (I/R, n = 6), I/R animals treated with biotinylated catalase (CAT, n = 6), and I/R rats treated with the conjugates (9B9-CAT, n = 6). The 9B9-CAT accumulation in the pulmonary endothelium of injured lungs was elucidated immunohistochemically. Arterial oxygenation during reperfusion was significantly higher in 9B9-CAT (221 ± 36 mmHg) and sham (215 ± 16 mmHg; P < 0.001 for both) compared with I/R (110 ± 10 mmHg) and CAT (114 ± 30 mmHg). Wet-dry weight ratio of I/R (6.78 ± 0.94%) and CAT (6.54 ± 0.87%) was significantly higher than of sham (4.85 ± 0.29%; P < 0.05), which did not differ from 9B9-CAT (5.58 ± 0.80%). The significantly lower degree of lung injury in 9B9-CAT-treated animals compared with I/R rats was also shown by decreased serum levels of endothelin-1 (sham, 18 ± 9 fmol/mg; I/R, 42 ± 12 fmol/mg; CAT, 36 ± 11 fmol/mg; 9B9-CAT, 26 ± 9 fmol/mg; P < 0.01) and mRNA for inducible nitric oxide synthase (iNOS) [iNOS-GAPDH ratio: sham, 0.15 ± 0.06 arbitrary units (a.u.); I/R, 0.33 ± 0.08 a.u.; CAT, 0.26 ± 0.05 a.u.; 9B9-CAT, 0.14 ± 0.04 a.u.; P < 0.001]. These results validate immunotargeting by anti-ACE conjugates as a prospective and specific strategy to augment antioxidative defenses of the pulmonary endothelium in vivo.

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