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Richard C. Holz - One of the best experts on this subject based on the ideXlab platform.
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Identification of a Histidine Metal Ligand in the Arge-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli
SpringerPlus, 2013Co-Authors: Wade C. Mcgregor, Sabina I. Swierczek, Danuta M Gillner, Richard C. HolzAbstract:The H355A, H355K, H80A, and H80K mutant enzymes of the Arge -encoded N -acetyl-L-ornithine deacetylase (Arge) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT Arge. Electronic absorption spectra of Co(II)-loaded H355A-Arge indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant ( K _d) of 39 μM. A three-dimensional homology model of Arge was generated using the X-ray crystal structure of the dapE -encoded N -succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.
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Structural characterization of Zn(II)-, Co(II)-, and Mn(II)-loaded forms of the Arge-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli.
Journal of inorganic biochemistry, 2012Co-Authors: Ye Tao, Jacob E. Shokes, Wade C. Mcgregor, Robert A. Scott, Richard C. HolzAbstract:The Zn, Co, and Mn K-edge extended X-ray absorption fine structure (EXAFS) spectra of the N-acetyl-l-ornithine deacetylase (Arge) from Escherichia coli, loaded with one or two equivalents of divalent metal ions (i.e., [Zn(II)_(Arge)], [Zn(II)Zn(II)(Arge)], [Co(II)_(Arge)], [Co(II)Co(II)(Arge)], [Mn(II)_(Arge)], and [Mn(II)Mn(II)(Arge)]), were recorded. The Fourier transformed data (FT) for [Zn(II)_(Arge)], [Zn(II)Zn(II)(Arge)], [Co(II)_(Arge)] and [Co(II)Co(II)(Arge)] are dominated by a peak at 2.05A, that can be fit assuming five or six light atom (N,O) scatterers. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f'). Furthermore, the data best fit a model that included a M-M vector at 3.3 and 3.4A for Zn(II) and Co(II), respectively, suggesting the formation of a dinuclear site. Multiple scattering contributions from the outer-shell atoms of a histidine-imidazole rings are observed at ~3 and 4A for Zn(II)- and Co(II)-loaded Arge suggesting at least one histidine ligand at each metal binding site. Likewise, EXAFS data for Mn(II)-loaded Arge are dominated by a peak at 2.19A that was best fit assuming six light atom (N,O) scatterers. Due to poor signal to noise ratios for the Mn EXAFS spectra, no Mn-Mn vector could be modeled. Peak intensities for [M(II)_(Arge)] vs. [M(II)M(II)(Arge)] suggest the Zn(II), Co(II), and Mn(II) bind to Arge in a cooperative manner. Since no structural data has been reported for any Arge enzyme, the EXAFS data reported herein represent the first structural glimpse for Arge enzymes. These data also provide a structural foundation for the future design of small molecules that function as inhibitors of Arge and may potentially function as a new class of antibiotics.
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Inhibitors of N ^α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency
Amino Acids, 2010Co-Authors: Jan Hlavacek, Jirina Slaninova, Donald Gilner, Vladimír Fučík, Jiri Jiracek, Jan Picha, V. Vaněk, M. Buděšínský, Richard C. HolzAbstract:A series of N ^α-acyl (alkyl)- and N ^α-alkoxycarbonyl-derivatives of l - and d -ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N ^α-acetyl- l -ornithine deacetylase (Arge). Arge catalyzes the conversion of N ^α-acetyl- l -ornithine to l -ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC_50 values in the μM range toward Arge, indicating that they are moderately strong inhibitors. N ^α-chloroacetyl- l -ornithine ( 1g ) was the best inhibitor tested toward Arge providing an IC_50 value of 85 μM while N ^α-trifluoroacetyl- l -ornithine ( 1f ), N ^α-ethoxycarbonyl- l -ornithine ( 2b ), and N ^α-acetyl- d -ornithine ( 1a ) weakly inhibited Arge activity providing IC_50 values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis- 168 for N ^α-acetyl- d -ornithine ( 1a ) and N ^α-fluoro- ( 1f ), N ^α-chloro- ( 1g ), N ^α-dichloro- ( 1h ), and N ^α-trichloroacetyl-ornithine ( 1i ) was also observed. These data correlate well with the IC_50 values determined for Arge, suggesting that these compounds might be capable of getting across the cell membrane and that Arge is likely the bacterial enzymatic tArget.
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Inhibitors of Nα -acetyl-l-ornithine Deacetylase: Synthesis, Characterization and Analysis of their Inhibitory Potency
Amino Acids, 2009Co-Authors: Jan Hlavacek, Václav Vaněk, Jirina Slaninova, Donald Gilner, Vladimír Fučík, Miloš Buděšínský, Jiri Jiracek, Jan Picha, Richard C. HolzAbstract:A series of N α-acyl (alkyl)- and N α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N α-acetyl-l-ornithine deacetylase (Arge). Arge catalyzes the conversion of N α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC50 values in the μM range toward Arge, indicating that they are moderately strong inhibitors. N α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward Arge providing an IC50 value of 85 μM while N α-trifluoroacetyl-l-ornithine (1f), N α-ethoxycarbonyl-l-ornithine (2b), and N α-acetyl-d-ornithine (1a) weakly inhibited Arge activity providing IC50 values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N α-acetyl-d-ornithine (1a) and N α-fluoro- (1f), N α-chloro- (1g), N α-dichloro- (1h), and N α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC50 values determined for Arge, suggesting that these compounds might be capable of getting across the cell membrane and that Arge is likely the bacterial enzymatic tArget.
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Characterization of the catalytically active Mn(II)-loaded Arge-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli
JBIC Journal of Biological Inorganic Chemistry, 2007Co-Authors: Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. HolzAbstract:The catalytically competent Mn(II)-loaded form of the Arge -encoded N -acetyl- l -ornithine deacetylase from Escherichia coli (Arge) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N -acetyl- l -ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k _cat and K _m values of 550 s^−1 and 0.8 mM, respectively, providing a catalytic efficiency ( k _cat/ K _m) of 6.9 × 10^5 M^−1 s^−1. The Arge dissociation constant ( K _d) for Mn(II) was determined to be 0.18 μM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 μM for the first metal binding event and 5.3 μM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded Arge was linear from 15 to 55 °C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol^−1. Other thermodynamic parameters were Δ G ^‡ = 58.1 kJ mol^−1, Δ H ^‡ = 47.7 kJ mol^−1, and Δ S ^‡ = –34.5 J mol^−1 K^−1. Similarly, plots of ln K _m versus 1/ T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2 S ,3 R )-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of Arge with a K _i value of 67 μM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(Arge)] and [Mn(II)Mn(II)(Arge)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(Arge)] in the presence of bestatin indicates that bestatin binds to Arge but does not form a µ-alkoxide bridge between the two metal ions.
Carrie Haskellluevano - One of the best experts on this subject based on the ideXlab platform.
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synergistic multiresidue substitutions of a macrocyclic c pro arg phe phe asn ala phe dpro agouti related protein agrp scaffold yield potent and 600 fold mc4r versus mc3r selective melanocortin receptor antagonists
Journal of Medicinal Chemistry, 2018Co-Authors: Katlyn A Fleming, Katie T Freeman, Mark D Ericson, Carrie HaskellluevanoAbstract:Antagonist ligands of the melanocortin-3 and -4 receptors (MC3R, MC4R), including agouti-related protein (AGRP), are postulated to be tArgets for the treatment of diseases of negative energy balance. Previous studies reported the macrocyclic MC3R/MC4R antagonist c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-dPro8], which is 250-fold less potent at the mouse (m) mMC3R and 3-fold less potent at the mMC4R than AGRP. Previous studies explored the structure–activity relationships around individual positions in this template. Herein, a multiresidue substitution strategy is utilized, combining the lead sequence with hPhe4, Dap5, Arg5, Ser6, and Nle7 substitutions previously reported. Two compounds from this study (16, 20) contain an hPhe4/Ser6/Nle7 substitution pattern, are 3–6-fold more potent than AGRP at the mMC4R and are 600–800-fold selective for the mMC4R over the mMC3R. Another lead compound (21), possessing the hPhe4/Arg5 substitutions, is only 5-fold less potent than AGRP at the mMC3R and is equipotent to AGRP a...
Katlyn A Fleming - One of the best experts on this subject based on the ideXlab platform.
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synergistic multiresidue substitutions of a macrocyclic c pro arg phe phe asn ala phe dpro agouti related protein agrp scaffold yield potent and 600 fold mc4r versus mc3r selective melanocortin receptor antagonists
Journal of Medicinal Chemistry, 2018Co-Authors: Katlyn A Fleming, Katie T Freeman, Mark D Ericson, Carrie HaskellluevanoAbstract:Antagonist ligands of the melanocortin-3 and -4 receptors (MC3R, MC4R), including agouti-related protein (AGRP), are postulated to be tArgets for the treatment of diseases of negative energy balance. Previous studies reported the macrocyclic MC3R/MC4R antagonist c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-dPro8], which is 250-fold less potent at the mouse (m) mMC3R and 3-fold less potent at the mMC4R than AGRP. Previous studies explored the structure–activity relationships around individual positions in this template. Herein, a multiresidue substitution strategy is utilized, combining the lead sequence with hPhe4, Dap5, Arg5, Ser6, and Nle7 substitutions previously reported. Two compounds from this study (16, 20) contain an hPhe4/Ser6/Nle7 substitution pattern, are 3–6-fold more potent than AGRP at the mMC4R and are 600–800-fold selective for the mMC4R over the mMC3R. Another lead compound (21), possessing the hPhe4/Arg5 substitutions, is only 5-fold less potent than AGRP at the mMC3R and is equipotent to AGRP a...
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Synergistic Multiresidue Substitutions of a Macrocyclic c[Pro-Arg-Phe-Phe-Asn-Ala-Phe‑dPro] Agouti-Related Protein (AGRP) Scaffold Yield Potent and >600-Fold MC4R versus MC3R Selective Melanocortin Receptor Antagonists
2018Co-Authors: Katlyn A Fleming, Katie T Freeman, Mark D Ericson, Carrie Haskell-luevanoAbstract:Antagonist ligands of the melanocortin-3 and -4 receptors (MC3R, MC4R), including agouti-related protein (AGRP), are postulated to be tArgets for the treatment of diseases of negative energy balance. Previous studies reported the macrocyclic MC3R/MC4R antagonist c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-dPro8], which is 250-fold less potent at the mouse (m) mMC3R and 3-fold less potent at the mMC4R than AGRP. Previous studies explored the structure–activity relationships around individual positions in this template. Herein, a multiresidue substitution strategy is utilized, combining the lead sequence with hPhe4, Dap5, Arg5, Ser6, and Nle7 substitutions previously reported. Two compounds from this study (16, 20) contain an hPhe4/Ser6/Nle7 substitution pattern, are 3–6-fold more potent than AGRP at the mMC4R and are 600–800-fold selective for the mMC4R over the mMC3R. Another lead compound (21), possessing the hPhe4/Arg5 substitutions, is only 5-fold less potent than AGRP at the mMC3R and is equipotent to AGRP at the mMC4R
Kentaro Takayama - One of the best experts on this subject based on the ideXlab platform.
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discovery of a human neuromedin u receptor 1 selective hexapeptide agonist with enhanced serum stability
Journal of Medicinal Chemistry, 2017Co-Authors: Kentaro Takayama, Miwa Mori, Akiko Tanaka, Erina Nomura, Yuko Sohma, Akihiro Taguchi, Atsuhiko Taniguchi, Kenji Mori, Toshiyasu Sakane, Akira YamamotoAbstract:Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp1-Phe(4-F)2-Arg3-Pro4-Arg5-Asn6-NH2 (4). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure–activity relationship study focused on residue 2 of agonist 4, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist 7b that suppresses body weight gain in mice.
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Discovery of a Human Neuromedin U Receptor 1‑Selective Hexapeptide Agonist with Enhanced Serum Stability
2017Co-Authors: Kentaro Takayama, Miwa Mori, Akiko Tanaka, Erina Nomura, Yuko Sohma, Akihiro Taguchi, Atsuhiko Taniguchi, Kenji Mori, Toshiyasu Sakane, Akira YamamotoAbstract:Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp1-Phe(4-F)2-Arg3-Pro4-Arg5-Asn6-NH2 (4). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure–activity relationship study focused on residue 2 of agonist 4, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist 7b that suppresses body weight gain in mice
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discovery of potent hexapeptide agonists to human neuromedin u receptor 1 and identification of their serum metabolites
ACS Medicinal Chemistry Letters, 2015Co-Authors: Kentaro Takayama, Yuko Sohma, Akihiro Taguchi, Kenji Mori, Koji Taketa, Fumika Yakushiji, Naoto Minamino, Mikiya Miyazato, Kenji Kangawa, Yoshio HayashiAbstract:Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists 2 and 3, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure–activity relationship study, which led to the identification of the more potent hexapeptide 5d that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including 5d, in rat serum, and identified two major biodegradation sites: Phe2-Arg3 and Arg5-Asn6. The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in 5d, enhanced the metabolic stability at Phe2-Arg3. These results pro...
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Discovery of Potent Hexapeptide Agonists to Human Neuromedin U Receptor 1 and Identification of Their Serum Metabolites
2015Co-Authors: Kentaro Takayama, Yuko Sohma, Akihiro Taguchi, Kenji Mori, Koji Taketa, Fumika Yakushiji, Naoto Minamino, Mikiya Miyazato, Kenji Kangawa, Yoshio HayashiAbstract:Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists 2 and 3, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure–activity relationship study, which led to the identification of the more potent hexapeptide 5d that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including 5d, in rat serum, and identified two major biodegradation sites: Phe2-Arg3 and Arg5-Asn6. The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in 5d, enhanced the metabolic stability at Phe2-Arg3. These results provide important information to guide the development of practical hNMU agonists
Patrick E. Ward - One of the best experts on this subject based on the ideXlab platform.
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depressor action of bradykinin agonists relative to metabolism by angiotensin converting enzyme carboxypeptidase n and aminopeptidase p
Experimental Biology and Medicine, 1992Co-Authors: Saleem Ahmad, Patrick E. WardAbstract:AbstractBradykinin (BK) receptor agonists and antagonists contain modifications that confer resistance to specific peptidases. In control studies, rat plasma degraded BK (10.3 ± 0.3 nmol/min/ml) via angiotensin-converting enzyme (ACE; EC 3.4.15.1; 5.2 ± 0.3 nmol/min/ml), carboxypeptidase N (CPN; EC 3.4.17.3; 3.2 ± 0.4 nmol/min/ml), aminopeptidase P (APP; EC 3.4.11.9; 0.6 ± 0.2 nmol/min/ml), and other (unidentified) activity (2.1 ± 0.6 nmol/min/ml). In contrast, BK agonist analogs were hydrolyzed more slowly due to selective resistance to these plasma peptidases. In addition to Lys-Lys-BK (B1087), which is partially resistant to ACE, [Hyp2,Phe8-r-Arg9]BK (B7642) was completely resistant to ACE, CPN, and the unidentified plasma activity (1.9 ± 0.3 nmol/min/ml), and D-Arg0[Hyp3,Phe8-r-Arg9]BK (B7644) was resistant to all plasma hydrolysis, including APP (<0.2 nmol/min/ml). In vivo ACE-resistant B1087 exhibited a depressor potency and duration of action greater than BK and equivalent to that of BK in the pres...
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Depressor action of bradykinin agonists relative to metabolism by angiotensin-converting enzyme, carboxypeptidase N, and aminopeptidase P.
Experimental Biology and Medicine, 1992Co-Authors: Saleem Ahmad, Patrick E. WardAbstract:AbstractBradykinin (BK) receptor agonists and antagonists contain modifications that confer resistance to specific peptidases. In control studies, rat plasma degraded BK (10.3 ± 0.3 nmol/min/ml) via angiotensin-converting enzyme (ACE; EC 3.4.15.1; 5.2 ± 0.3 nmol/min/ml), carboxypeptidase N (CPN; EC 3.4.17.3; 3.2 ± 0.4 nmol/min/ml), aminopeptidase P (APP; EC 3.4.11.9; 0.6 ± 0.2 nmol/min/ml), and other (unidentified) activity (2.1 ± 0.6 nmol/min/ml). In contrast, BK agonist analogs were hydrolyzed more slowly due to selective resistance to these plasma peptidases. In addition to Lys-Lys-BK (B1087), which is partially resistant to ACE, [Hyp2,Phe8-r-Arg9]BK (B7642) was completely resistant to ACE, CPN, and the unidentified plasma activity (1.9 ± 0.3 nmol/min/ml), and D-Arg0[Hyp3,Phe8-r-Arg9]BK (B7644) was resistant to all plasma hydrolysis, including APP (
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metabolism of bradykinin agonists and antagonists by plasma aminopeptidase p
Biochemical Pharmacology, 1991Co-Authors: Patrick E. Ward, Ana Chow, Guy DrapeauAbstract:Abstract In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1–5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L -mercapto-methyl-3-guanidino-ethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)]BK and [Phe8ψ(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [ D -Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for B K (Km = 19.7 ± 2.6 μM; Vmax = 12.1 ± 1.2 nmol/min/mL). In contrast, B2 antagonists containing D -Arg0 N-termini, including D -Arg[Hyp3,Thi5, 8, D -Phe7]BK and D -Arg[Hyp3, D -Phe7, Phe8ψ(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D -Arg0-containing antagonists.
