Rapid radioassay for prolylcarboxypeptidase (angiotensinase C)

Skidgel, Randal A.; Wickstrom, Eric; Kumamoto, Kenshi; Erdös, Ervin G.

doi:10.1016/0003-2697(81)90165-2

Cited by 25 publications

(14 citation statements)

References 13 publications

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“…N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) was obtained from Sigma and Peptides International (Louisville, KY). N-Benzyloxycarbonyl-L-prolyl-L-[3H]alanine (N-Cbz-Pro-[3H]Ala) was prepared as described (11). Bestatin and phosphoramidon were obtained from Peninsula Laboratories (Belmont, CA).…”

Section: Methodsmentioning

confidence: 99%

Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.

Connelly¹,

Skidgel²,

Schulz³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

156

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Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-AlaPhe-MNA) at a rate of 9.5 nmol hr-1 per 106 cells, and phosphoramidon (1 IAM) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA (96 nmol min-'-mg-1) and the hydrolysis was inhibited by phosphoramidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol mini1mgi1) and 0.1 mM fMet-Leu-Phe (49 nmol min-1 mg 1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (K., 59 x 10-6 M; k~,tq 3654 min-'). Immunocytochemistry at the light microscopic level revealed a high concentration of NEP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.

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Section: Methodsmentioning

confidence: 99%

Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.

Connelly¹,

Skidgel²,

Schulz³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

156

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“…Antigen-affinity purified goat antihuman SDF-1␣ and ␤ (BAF-310), and biotin-labeled mouse immunoglobulin G 1 (IgG1) monoclonal anti-human/mouse SDF-1 antibodies (MAB 310) were from R&D Systems; rabbit anti-human SDF-1 antigen affinitypurified antibodies were from PeproTech (Rocky Hill, NJ). Anti-CPN immune serum 30 was a kind gift of Dr E. G. Erdös (UIC College of Medicine, Chicago, IL). Purified CPN was a kind gift of Dr Mariko Nagashima, Berlex Biosciences (Richmond, CA).…”

Section: Reagents and Samplesmentioning

confidence: 99%

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1α in the circulation

Davis

Singer

Sierra

et al. 2005

Blood

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The chemokine stromal-derived factor-1␣ (SDF-1␣) is an essential regulator of hematopoiesis, lymphocyte homing, pre-Bcell growth, and angiogenesis. As SDF-1␣ is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1␣, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1␣, leading to significant reduction in biologic activity, have not been identified.Using a new biochemical assay for measuring the carboxy-terminal cleavage activity, we purified from serum and plasma a peptidase that specifically removes the carboxy-terminal lysine from SDF-1␣ and identified it as carboxypeptidase N (CPN, also known as kininase I, arginine carboxypeptidase, and anaphylotoxin inactivator). We demonstrate that SDF-1␣ in serum and plasma lacks the carboxy terminal lysine, and depletion of CPN from serum and plasma significantly reduces the SDF-1␣ carboxypeptidase activity. Purified CPN effectively and specifically removes the carboxy-terminal lysine from SDF-1␣ and significantly reduces the chemokine's biologic activity as a pre-B-cell growth factor and chemoattractant. Thus, in addition to its role as a regulator of the biologic activity of kinins and anaphylatoxins, CPN is an important regulator of the biologic activity of SDF-1␣ by reducing the chemokine-specific activity. IntroductionThe chemokine stromal-derived factor-1 (SDF-1) and its unique receptor, CXCR4, are key regulators of the hematopoietic, nervous, and cardiovascular systems during embryogenesis. 1-3 Knock-out mice for SDF-1 or CXCR4 die perinatally with a similar phenotype, including defects in the formation of the cerebellum, the ventricular septum of the heart, the vasculature of the gastrointestinal system, and the lympho-hematopoietic system. [4][5][6][7] After birth, SDF-1 and CXCR4 continue to play key roles in regulating physiologic hematopoiesis and angiogenesis. [8][9][10][11][12][13][14][15] Studies in vitro have shown that SDF-1 functions as a growth factor for immature B lymphocytes and promotes chemotaxis in CXCR4-expressing cells, including tumor cells. 1,3,9,[16][17][18] Since CXCR4 can function as a coreceptor for T-cell tropic HIV-1, SDF-1 and CXCR4 may also affect the course of HIV-1 infection. 19,20 SDF-1␣ is a highly conserved chemokine in evolution, only a single conservative substitution (I to V) distinguishes the human chemokine from the mouse chemokine. 21,22 Two forms of SDF-1 are generated, ␣ and ␤, which are identical in amino acid sequence except for the presence of 4 additional amino acids at the carboxy terminus of SDF-1␤. 21,22 A third form of SDF-1 was identified in rat, SDF-1␥, which is predicted to code for a protein identical to SDF-1␤, except for the insertion of 30 additional amino acids at the carboxy terminus. 23 Structure-function studies have identified the amino terminal region of SDF-1 as critical to CXCR4 receptor activation. 22,24 SDF-1 lacking the amino term...

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“…These conditions were met by the radioassay described here. It was designed following the basic principles of an assay for prolyl-carboxypeptidase (angiotensinase C) (Skidgel et al, 1981) except that the radioactive peptide was coupled to agarose to facilitate the separation of the cleavage product from the substrate. This method was successfully employed as an assay not only for the virus-associated enzyme, but also for pancreatic carboxypeptidase B (Fig.…”

Section: Is the Carboxypeptidase Essential For The Activation Of The mentioning

confidence: 99%

Characterization of the Carboxypeptidase Involved in the Proteolytic Cleavage of the Influenza Haemagglutinin

Garten

Klenk

1983

Journal of General Virology

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SUMMARYThe arginine carboxypeptidase involved in the proteolytic cleavage of the haemagglutinin of influenza A virus has been analysed by an assay employing a Sepharose-bound peptide containing radioactive arginine as a substrate. The enzyme activity has been extracted from purified virus with non-ionic detergents and has been separated from the haemagglutinin and from the neuraminidase by isoelectric focusing and by affinity chromatography. The carboxypeptidase present in virus grown in different host cells shows variations in its isoelectric point. It can be concluded from these observations that the carboxypeptidase is a host component incorporated into the virus envelope. When the enzyme is inhibited by 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, haemagglutinin with the arginine attached to the carboxy terminus of HA~ can be obtained. The observation that under these conditions the haemagglutinin has retained its haemolytic activity indicates that the carboxypeptidase does not play an essential role in the activation process.

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Rapid radioassay for prolylcarboxypeptidase (angiotensinase C)

Cited by 25 publications

References 13 publications

Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.

Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1α in the circulation

Characterization of the Carboxypeptidase Involved in the Proteolytic Cleavage of the Influenza Haemagglutinin

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