Journal articles: 'Carboxypeptidase A'

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Fan, Xuemo, Sandy J. Olson, Lewis S. Blevins, George S. Allen, and Mahlon D. Johnson. "Immunohistochemical Localization of Carboxypeptidases D, E, and Z in Pituitary Adenomas and Normal Human Pituitary." Journal of Histochemistry & Cytochemistry 50, no. 11 (November 2002): 1509–15. http://dx.doi.org/10.1177/002215540205001111.

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Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas.

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Greene, D., B. Das, and L. D. Fricker. "Regulation of carboxypeptidase E. Effect of pH, temperature and Co2+ on kinetic parameters of substrate hydrolysis." Biochemical Journal 285, no. 2 (July 15, 1992): 613–18. http://dx.doi.org/10.1042/bj2850613.

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Carboxypeptidase E is a member of the carboxypeptidase A and B gene family, with many of the putative active-site and substrate-binding residues conserved between these enzymes. However, the pH optimum of carboxypeptidase E is substantially lower than that of carboxypeptidases A and B. To evaluate whether the difference in the pH optima of these carboxypeptidases reflects fundamental differences in the ionization behaviour of active-site residues, the influence of pH on carboxypeptidase E activity was examined. The V(max) for hydrolysis of dansyl-Phe-Ala-Arg is pH-independent between 5 and 7, but decreases at pH values below 5. The pKa for the group the protonation of which leads to the loss of activity is approximately 4.8, and the slope of the V(max.)/pH profile suggests that only a single ionizable group is involved. In contrast, Km and V(max.)/Km are dramatically influenced by pH over the range 5-7, with multiple ionizable groups detected in this pH range. The pKa of the group the protonation of which decreases the V(max.) of substrate hydrolysis is lower (4.5) for carboxypeptidase E which had been reconstituted with Co2+. The enthalpy of ionization of the group observed in the V(max.) profile for carboxypeptidase E is approx. 28.9 kJ/mol. These results are compatible with the active-site model of the homologous carboxypeptidase A: in this model the ionization of a metal-bound water molecule is responsible for the observed decrease in V(max.).

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Deddish, P. A., R. A. Skidgel, and E. G. Erdös. "Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase)." Biochemical Journal 261, no. 1 (July 1, 1989): 289–91. http://dx.doi.org/10.1042/bj2610289.

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Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.

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Vitols, Karin S., Yolanda D. Montejano, Ulrike Kuefner, and F. M. Huennekens. "Selective Cytotoxicity of Carboxypeptidase-activated Methotrexate ex-Peptides." Pteridines 1, no. 1 (February 1989): 65–69. http://dx.doi.org/10.1515/pteridines.1989.1.1.65.

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Summary Methotrexate ex-pep tides (derivatives in which an amino acid is linked covalently to the ex-carboxyl of the glutamate residue on the parent drug) can be hydrolyzed by specific carboxypeptidases to yield free Methotrexate (MTX) and the corresponding amino acid. Studies with L12l0 cells in suspension culture have shown that the MTX pep tides can serve as "pro-drugs": Because of their inability to be taken up by cells, they are relatively non-toxic. When co-administered with appropriate carboxypeptidases, however, they become equitoxic with MTX. In the present investigation, the potential of the MTX-ala/carboxypeptidase A combination for providing regional cytotoxicity was demonstrated in a model system involving L12l0 cells propagated in semi-solid agarose. When cells and one of the components (MTX peptide or carboxypeptidase) were distributed uniformly throughout the agarose, and the other component was immobilized at the center, a discrete zone of cell kill radiated from the fixed component. These results suggest the possibility of developing a new mode of cancer chemotherapy involving circulating MTX peptides in conjunction with carboxypeptidases linked covalently to tumor-targeted monoclonal antibodies.

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Kimura, Yoshio, Yukie Takashima, Yushi Tokumasu, and Masayuki Sato. "Molecular Cloning, Sequence Analysis, and Characterization of a Penicillin-Resistantdd-Carboxypeptidase of Myxococcus xanthus." Journal of Bacteriology 181, no. 15 (August 1, 1999): 4696–99. http://dx.doi.org/10.1128/jb.181.15.4696-4699.1999.

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ABSTRACT We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistantdd-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity toN-acetylmuramoyl-l-alanine amidase and penicillin-resistant dd-carboxypeptidase, respectively. ThepdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar to those ofdd-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in pdcA deletion mutants.

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Ishikawa, Kazuhiko, Hiroyasu Ishida, Ikuo Matsui, Yutaka Kawarabayasi, and Hisasi Kikuchi. "Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3." Applied and Environmental Microbiology 67, no. 2 (February 1, 2001): 673–79. http://dx.doi.org/10.1128/aem.67.2.673-679.2001.

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ABSTRACT Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase ofSulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

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TAN, Fulong, Michael REHLI, Stefan W. KRAUSE, and Randal A. SKIDGEL. "Sequence of human carboxypeptidase D reveals it to be a member of the regulatory carboxypeptidase family with three tandem active site domains." Biochemical Journal 327, no. 1 (October 1, 1997): 81–87. http://dx.doi.org/10.1042/bj3270081.

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We have cloned the cDNA for human carboxypeptidase D (CPD), a new B-type metallocarboxypeptidase that is membrane bound and has an acidic pH optimum. The 5.8 kb of cDNA sequenced contains an open reading frame of 4131 bp encoding 1377 amino acid residues. The sequence is similar (75% identity) to duck gp180, a protein that was isolated, cloned and sequenced as a hepatitis B virus-binding protein but not characterized as a carboxypeptidase. Hydropathic analysis revealed a hydrophobic region at the N-terminus, representing the signal peptide, and one near the C-terminus that probably represents the transmembrane anchor. The most striking feature is the presence of three tandem carboxypeptidase homology domains that have sequence similarity to the regulatory B-type carboxypeptidase family, typified by carboxypeptidases M, E and N. Because of the three repeats, CPD is about three times larger (175–180 kDa) than other members of this family (approx. 50–62 kDa). Domain 2 is most closely related to carboxypeptidases M, E and N (45–48% identity), followed by domain 1 (37–38%) and domain 3 (20–27%). There is much higher sequence identity in regions containing putative active site residues, and all catalytically important residues are strictly conserved in domains 1 and 2. In domain 3, however, only 1 of 8 active site residues is conserved, indicating that this portion might not be catalytically active. Northern blotting of mRNA from human tissues and cells showed high levels of CPD mRNA in placenta, pancreas and Hep G2 hepatoma cells, and smaller amounts in skeletal muscle, heart and HT-29 colon carcinoma and melanoma cell lines.

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Vilanova, M., J. Vendrell, M. T. López, C. M. Cuchillo, and F. X. Avilés. "Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A." Biochemical Journal 229, no. 3 (August 1, 1985): 605–9. http://dx.doi.org/10.1042/bj2290605.

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A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods. Undegraded and homogeneous carboxypeptidase A1 and A2 species (peptidyl-L-amino acid hydrolase, EC 3.4.17.1), in monomeric forms with high specific activity, are also obtained in high yield by controlled trypsin activation of either of the pro-(carboxypeptidases A) followed by chromatography on DEAE-Sepharose at pH 5.8 under dissociating conditions (7 M-urea).

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Masuda, Kaname, Masami Yoshioka, Daisuke Hinode, and Ryo Nakamura. "Purification and Characterization of Arginine Carboxypeptidase Produced by Porphyromonas gingivalis." Infection and Immunity 70, no. 4 (April 2002): 1807–15. http://dx.doi.org/10.1128/iai.70.4.1807-1815.2002.

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ABSTRACT Arginine carboxypeptidase was isolated from the cytoplasm of Porphyromonas gingivalis 381 and purified by DEAE-Sephacel column chromatography, followed by high-performance liquid chromatography on DEAE-5PW and TSK G2000SWXL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of three major bands at 42, 33, and 32 kDa with identical N-terminal sequences. By Western blotting analysis and immunoelectron microscopy, the arginine carboxypeptidase was found to be widely distributed in the cytoplasm and on the surface of the outer membrane. The open reading frame corresponding to the N-terminal amino acids of the arginine carboxypeptidase was detected by a search of the sequence of the P. gingivalis W83 genome. This sequence showed homology with mammalian carboxypeptidases (M, N, and E/H) and included a zinc-binding region signature, suggesting that the enzyme is a member of the zinc carboxypeptidase family. The purified enzyme was inhibited by EGTA, o-phenanthroline, dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and some metal ions, such as Cu2+, Zn2+, and Cd2+. On the other hand, Co2+ activated the enzyme. The enzyme released arginine and/or lysine from biologically active peptides containing these amino acids at the C terminus but did not cleave substrates when proline was present at the penultimate position. These results indicate that the arginine carboxypeptidase produced by P. gingivalis is an exo type of metallocarboxypeptidase. This enzyme may function to release arginine in collaboration with an arginine aminopeptidase, e.g., Arg-gingipain, to obtain specific amino acids from host tissues during the growth of P. gingivalis.

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Shevchenko, K. V., L. A. Andreeva, I. Yu Nagaev, V. P. Shevchenko, and N. F. Myasoedov. "Study of stability of proline-containing derivatives of dopamine and serotonin in the biological media in vitro experiments." Biomeditsinskaya Khimiya 65, no. 6 (2019): 498–506. http://dx.doi.org/10.18097/pbmc20196506498.

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Boc-Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt were synthesized for the first time. The stability of these compounds in the presence of leucine aminopeptidase, carboxypeptidase Y, carboxypeptidase B and proline endopeptidase (PEP) was determined. It turned out that the compounds are stable in the presence of aminopeptidases and carboxypeptidases. In the presence of PEP, dopamine (DP) and serotonin (Srt) are cleaved from the synthesized preparations. Thus, new proline-containing Srt and DP derivatives were obtained, Srt and DP could be gradually released from them. This suggest the possibility of a prolonged action of these biologically active compounds on the vital activity of cells and, consequently, of the whole organism.

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Watanabe, T. "Proteolytic Activities of Mycoplasma Salivarium." Advances in Dental Research 2, no. 2 (November 1988): 297–300. http://dx.doi.org/10.1177/08959374880020021501.

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Proteolytic activity was demonstrated in ATCC 23064 and oral isolates (more than 100 strains) of My coplasma salivarium and was suggested to be due to aminopeptidase, some other metalloproteinases, and serine proteinases. An aminopeptidase and a carboxypeptidase were purified from cell membranes of the organism and characterized. In amino acid specificities, the aminopeptidase was different from aminopeptidase B and leucine aminopeptidase, and the carboxypeptidase from carboxypeptidases B and N. These enzymes are considered to play a role in providing the organism with arginine as a main energy source, eventually facilitating the production of ammonia, and in protecting the organism and the other co-existing pathogens from defense mechanisms of the host by inactivating kallidin, bradykinin, tuftsin, and anaphylatoxins such as C3a and C5a.

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Sangsree, Sakonwun, Viktor Brovkovych, Richard D. Minshall, and Randal A. Skidgel. "Kininase I-type carboxypeptidases enhance nitric oxide production in endothelial cells by generating bradykinin B1receptor agonists." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 6 (June 1, 2003): H1959—H1968. http://dx.doi.org/10.1152/ajpheart.00036.2003.

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Kininase I-type carboxypeptidases convert native kinin agonists for B2receptors into B1receptor agonists by specifically removing the COOH-terminal Arg residue. The membrane localization of carboxypeptidase M (CPM) and carboxypeptidase D (CPD) make them ideally situated to regulate kinin activity. Nitric oxide (NO) release from human lung microvascular endothelial cells (HLMVEC) was measured directly in real time with a porphyrinic microsensor. Bradykinin (1–100 nM) elicited a transient (5 min) peak of generation of NO that was blocked by the B2antagonist HOE 140, whereas B1agonist des-Arg10-kallidin caused a small linear increase in NO over 20 min. Treatment of HLMVEC with 5 ng/ml interleukin-1β and 200 U/ml interferon-γ for 16 h upregulated B1receptors as shown by an approximately fourfold increase in prolonged (>20 min) output of NO in response to des-Arg10-kallidin, which was blocked by the B1antagonist des-Arg10-Leu9-kallidin. B2receptor agonists bradykinin or kallidin also generated prolonged NO production in treated HLMVEC, which was significantly reduced by either a B1antagonist or carboxypeptidase inhibitor, and completely abolished with a combination of B1and B2receptor antagonists. Furthermore, CPM and CPD activities were increased about twofold in membrane fractions of HLMVEC treated with interleukin-1β and interferon-γ compared with control cells. Immunostaining localized CPD primarily in a perinuclear/Golgi region, whereas CPM was on the cell membrane. These data show that cellular kininase I-type carboxypeptidases can enhance kinin signaling and NO production by converting B2agonists to B1agonists, especially in inflammatory conditions.

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Morlot, Cécile, Lucile Pernot, Audrey Le Gouellec, Anne Marie Di Guilmi, Thierry Vernet, Otto Dideberg, and Andréa Dessen. "Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) fromStreptococcus pneumoniae." Journal of Biological Chemistry 280, no. 16 (December 13, 2004): 15984–91. http://dx.doi.org/10.1074/jbc.m408446200.

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Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The singled-Ala,d-Ala (d,d)-carboxypeptidase inStreptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. PBP3 folds into an NH2-terminal,d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated β-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel β-sheet surrounded by α-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (kcat/Km= 50,500m–1·s–1), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to “skid” on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.

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Hadkar, Vaishali, Sakonwun Sangsree, Stephen M. Vogel, Viktor Brovkovych, and Randal A. Skidgel. "Carboxypeptidase-mediated enhancement of nitric oxide production in rat lungs and microvascular endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 287, no. 1 (July 2004): L35—L45. http://dx.doi.org/10.1152/ajplung.00346.2003.

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Membrane-bound regulatory carboxypeptidases cleave only COOH-terminal basic residues from peptides and proteins. To investigate whether carboxypeptidase-generated arginine can increase nitric oxide (NO) synthesis we perfused rat lungs from animals challenged with LPS or used rat lung microvascular endothelial cells (RLMVEC) stimulated with LPS and IFN-γ, conditions that induced inducible NO synthase (iNOS) expression. Addition of carboxypeptidase substrate furylacryloyl-Ala-Arg (Fa-A-R) or Arg to the lung perfusate increased NO production two- to threefold. The carboxypeptidase inhibitor 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) blocked the effect of Fa-A-R but not free Arg. Lysine, an Arg transport inhibitor, blocked the increase in NO stimulated by Fa-A-R. HPLC analysis showed that Fa-A-R hydrolysis was blocked by MGTA but not lysine. In cytokine-treated RLMVEC, Fa-A-R also stimulated NO production inhibited by MGTA or lysine. Membrane fractions from rat lungs or RLMVEC contained carboxypeptidase M-like activity at neutral pH that increased twofold in RLMVEC treated with LPS + IFN-γ. The kinetics of NO production in RLMVEC was measured with a porphyrinic microsensor. Addition of 1 mM Arg or Fa-A-R to cells preincubated in Arg-free medium resulted in a slowly rising, prolonged (>20 min) NO output. NO production stimulated by Fa-A-R was blocked by MGTA or iNOS inhibitor 1400W. HPLC analysis of Fa-A-R hydrolysis revealed only 3.7 μM Arg was released over 20 min. Thus NO production in RLMVEC is stimulated more efficiently by Arg released from carboxypeptidase substrates than free Arg. These studies reveal a novel mechanism by which the Arg supply for NO production in inflammatory conditions may be maintained.

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Gifford, David J., Kevin A. Wenzel, and Doug L. Lammer. "Lodgepole pine seed germination. I. Changes in peptidase activity in the megagametophyte and embryonic axis." Canadian Journal of Botany 67, no. 9 (September 1, 1989): 2539–43. http://dx.doi.org/10.1139/b89-328.

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Lodgepole pine (Pinus contorta Dougl.) germination, as indicated by radicle emergence from the seed coat, was completed 2 days after imbibition at 28 °C. By this time, the hydrolysis of the megagametophyte storage protein reserve had commenced. This was 90% complete 5 days after imbibition. Megagametophytes and embryonic axes contained two classes of peptidases: aminopeptidases that hydrolyzed amino acid β-naphthylamides, and carboxypeptidases. Cell-free extract studies showed that carboxypeptidase activity was very low in mature seed megagametophytes and embryonic axes, but increased rapidly in these tissues after radicle emergence. However, maximum rates of carboxypeptidase activity only occurred when 70% of the megagametophyte storage protein reserve had been depleted 5 days after imbibition. Thus, it was unlikely that carboxypeptidases played a significant role in the rapid mobilization of megagametophyte storage proteins. Mature seed megagametophytes contained high levels of aminopeptidase activity. Following germination this level of activity was maintained in megagametophytes and only declined when storage protein hydrolysis was almost completed, 5 days after imbibition. It is proposed that aminopeptidases are involved in the hydrolysis of megagametophyte storage proteins in the germinated lodgepole pine seed.

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Campbell, F. C., Jon G. Houseman, and P. E. Morrison. "A preliminary characterization of alkaline digestive proteases in the posterior midgut of the face fly, Musca autumnalis De Geer." Canadian Journal of Zoology 65, no. 3 (March 1, 1987): 635–39. http://dx.doi.org/10.1139/z87-099.

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Alkaline proteases in the posterior midgut of the face fly, Musca autumnalis De Geer, were identified using specific synthetic substrates and nonspecific substrate azocasein. Identification was confirmed with potential protease activators and inhibitors. Optimal azocasein hydrolysis occurred at pH 8.0 and substrate hydrolysis was inhibited by EGTA (ethyleneglycol-bis-(2-aminoethyl ether)-N,N-tetraacetic acid), tosyl-L-lysine chloromethyl ketone (TLCK), and soybean trypsin inhibitor (STI). Tryptic proteolysis was identified by maximal hydrolysis, at pH 8.0, of trypsin specific substrates BAPNA (benzoyl-DL-arginine-p-nitroanilide) and TAME (tosyl-L-arginine methyl ester). TAME hydrolysis was inhibited by PMSF (phenylmethylsulfonyl fluoride), STI, pepstatin A, and dithiothreitol (DTT), but BAPNA hydrolysis was inhibited only by STI and DTT. Chymotrypsin was demonstrated by maximal hydrolysis of BTEE (benzoyl-L-tyrosine ethyl ester) at pH 8.0 and DTT, PMSF, and STI inhibited hydrolysis of this substrate. Aminopeptidase activity was demonstrated by maximal hydrolysis of leucine-p-nitro-anilide at pH 8.0 and the peptidase was inhibited by o-phenanthroline. Carboxypeptidase A-like enzyme hydrolyzed hippuryl-DL-phenyllactic acid maximally at pH 7.5 and carboxypeptidase B showed maximum hydrolysis of hippuryl-L-arginine at pH 7.0. Both carboxypeptidases were inhibited by EDTA (ethylene diamine tetraacetic acid), EGTA, and DTT and carboxypeptidase A was also inhibited by PMSF.

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Turner, Anthony J., Sarah R. Tipnis, Jodie L. Guy, Gillian I. Rice, and Nigel M. Hooper. "ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors." Canadian Journal of Physiology and Pharmacology 80, no. 4 (April 1, 2002): 346–53. http://dx.doi.org/10.1139/y02-021.

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A human zinc metalloprotease (termed ACEH or ACE2) with considerable homology to angiotensin- converting enzyme (ACE) (EC 3.4.15.1) has been identified and subsequently cloned and functionally expressed. The translated protein contains an N-terminal signal sequence, a single catalytic domain with zinc-binding motif (HEMGH), a transmembrane region, and a small C-terminal cytosolic domain. Unlike somatic ACE, ACEH functions as a carboxypeptidase when acting on angiotensin I and angiotensin II or other peptide substrates. ACEH may function in conjunction with ACE and neprilysin in novel pathways of angiotensin metabolism of physiological significance. In contrast with ACE, ACEH does not hydrolyse bradykinin and is not inhibited by typical ACE inhibitors. ACEH is unique among mammalian carboxypeptidases in containing an HEXXH zinc motif but, in this respect, resembles a bacterial enzyme, Thermus aquaticus (Taq) carboxypeptidase (EC 3.4.17.19). Collectrin, a developmentally regulated renal protein, is homologous with the C-terminal region of ACEH but has no similarity with ACE and no catalytic domain. Thus, the ACEH protein may have evolved as a chimera of a single ACE-like domain and a collectrin domain. The collectrin domain may regulate tissue response to injury whereas the catalytic domain is involved in peptide processing events.Key words: ACEH, ACE2, metalloprotease, collectrin, carboxypeptidase, angiotensin II.

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Dalle Ore, Florence, El Hassan Ajandouz, Thierry Giardina, and Antoine Puigserver. "The membrane-bound basic carboxypeptidase from hog intestinal mucosa1Enzymes: carboxypeptidase B (EC 3.4.17.2); carboxypeptidase D (EC 3.4.17.-); carboxypeptidase H (EC 3.4.17.10); carboxypeptidase M (EC 3.4.17.12); carboxypeptidase N (EC 3.4.17.3).1." Biochimica et Biophysica Acta (BBA) - Biomembranes 1421, no. 2 (October 1999): 234–48. http://dx.doi.org/10.1016/s0005-2736(99)00122-4.

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Grimwood, B. G., T. H. Plummer, and A. L. Tarentino. "Carboxypeptidase H." Journal of Biological Chemistry 264, no. 26 (September 1989): 15662–67. http://dx.doi.org/10.1016/s0021-9258(19)84883-4.

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Christianson, David W., and William N. Lipscomb. "Carboxypeptidase A." Accounts of Chemical Research 22, no. 2 (February 1989): 62–69. http://dx.doi.org/10.1021/ar00158a003.

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Fricker, L. D. "Carboxypeptidase E." Annual Review of Physiology 50, no. 1 (October 1988): 309–21. http://dx.doi.org/10.1146/annurev.ph.50.030188.001521.

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Cohen, Andrea J., Randal A. Skidgel, Laura B. Gilman, Judy K. Black, Paul A. Bunn, Barbara Helfrich, Wilbur A. Franklin, and York E. Miller. "Carboxypeptidase M." Chest 111, no. 6 (June 1997): 149S. http://dx.doi.org/10.1378/chest.111.6_supplement.149s.

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Schweisfurth, H. "Carboxypeptidase N." DMW - Deutsche Medizinische Wochenschrift 109, no. 33 (March 26, 2008): 1254–58. http://dx.doi.org/10.1055/s-2008-1069360.

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Rimsa, Vadim, Thomas C. Eadsforth, Robbie P. Joosten, and William N. Hunter. "High-resolution structure of the M14-type cytosolic carboxypeptidase fromBurkholderia cenocepaciarefined exploitingPDB_REDOstrategies." Acta Crystallographica Section D Biological Crystallography 70, no. 2 (January 29, 2014): 279–89. http://dx.doi.org/10.1107/s1399004713026801.

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A potential cytosolic metallocarboxypeptidase fromBurkholderia cenocepaciahas been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded inPDB_REDOwere coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. TheB. cenocepaciaprotein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

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Pfeifle, Dieter, Eva Janas, and Bernd Wiedemann. "Role of Penicillin-Binding Proteins in the Initiation of the AmpC β-Lactamase Expression inEnterobacter cloacae." Antimicrobial Agents and Chemotherapy 44, no. 1 (January 1, 2000): 169–72. http://dx.doi.org/10.1128/aac.44.1.169-172.2000.

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ABSTRACT Penicillin-binding proteins (PBPs) are involved in the regulation of β-lactamase expression by determining the level of anhydromuramylpeptides in the periplasmatic space. It was hypothesized that one or more PBPs act as a sensor in the β-lactamase induction pathway. We have performed induction studies with Escherichia coli mutants lacking one to four PBPs withdd-carboxypeptidase activity. Therefore, we conclude that a strong β-lactamase inducer must inhibit alldd-carboxypeptidases as well as the essential PBPs 1a, 1b, and/or 2.

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26

Chiba, Y., T. Midorikawa, and E. Ichishima. "Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis." Biochemical Journal 308, no. 2 (June 1, 1995): 405–9. http://dx.doi.org/10.1042/bj3080405.

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Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).

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27

Rigiani, N. R., R. A. Wevers, E. Rijk, and J. B. Soons. "Postsynthetic modification of human enolase isoenzymes." Clinical Chemistry 33, no. 6 (June 1, 1987): 757–60. http://dx.doi.org/10.1093/clinchem/33.6.757.

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Abstract The original form of beta beta enolase (EC 4.2.1.11) in tissue is modified to two more electrophoretically distinct forms when incubated with human serum. The three postsynthetic forms are designated beta beta 3, beta beta 2, and beta beta 1, in order of increasing anodal mobility and increasing modification. Serum and carboxypeptidases A and B all produce identical modifications of beta beta enolase but exhibit very different pH-activity profiles. A purified human serum protein previously named "modifying protein," which is responsible for the modification of creatine kinase-M and alpha-enolase subunits, modifies beta beta enolase and also has a pH-activity profile identical to that for serum. Thus we conclude that the modifying protein is not identical to either carboxypeptidase A or B; it may, however, be an as-yet-undescribed carboxypeptidase. With increased modification, both alpha alpha and beta beta enolase decrease in apparent activation energy; gamma gamma enolase shows no evidence of modification, and its apparent activation energy remains stable. Measurement of activation energy is an easy tool for screening for postsynthetic modifications in an enzyme.

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28

Skidgel, R. A., R. M. Davis, and F. Tan. "Human carboxypeptidase M." Journal of Biological Chemistry 264, no. 4 (February 1989): 2236–41. http://dx.doi.org/10.1016/s0021-9258(18)94167-0.

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29

Lewis, W. S., and S. M. Schuster. "Carboxypeptidase Y stability." Journal of Biological Chemistry 266, no. 31 (November 1991): 20818–22. http://dx.doi.org/10.1016/s0021-9258(18)54782-7.

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30

Redlitz, Alexander, Francesca A. Nicolini, Janis L. Malycky, Eric J. Topol, and Edward F. Plow. "Inducible Carboxypeptidase Activity." Circulation 93, no. 7 (April 1996): 1328–30. http://dx.doi.org/10.1161/01.cir.93.7.1328.

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31

Wang, Wei, Dirk F. Hendriks, and Simon L. Scharpé. "Immobilized carboxypeptidase N." Applied Biochemistry and Biotechnology 44, no. 2 (February 1994): 151–60. http://dx.doi.org/10.1007/bf02921652.

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32

Wang, W., D. F. Hendriks, and S. S. Scharpé. "Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen." Journal of Biological Chemistry 269, no. 22 (June 1994): 15937–44. http://dx.doi.org/10.1016/s0021-9258(17)40771-x.

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Hendriks, D., W. Wang, and S. Scharpe. "Carboxypeptidase U : A novel plasma carboxypeptidase with affinity for plasminogen." Fibrinolysis 8 (January 1994): 24. http://dx.doi.org/10.1016/0268-9499(94)90353-0.

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34

CONTIN, María A., Juan J. SIRONI, Héctor S. BARRA, and Carlos A. ARCE. "Association of tubulin carboxypeptidase with microtubules in living cells." Biochemical Journal 339, no. 2 (April 8, 1999): 463–71. http://dx.doi.org/10.1042/bj3390463.

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Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from α-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction. We ruled out, by using several control experiments, the possibility that this result was due to contamination of the isolated cytoskeletons by non-associated proteins contained in the detergent fraction or to an artifact in vitro during the extraction procedure. The associated carboxypeptidase activity showed characteristics similar to those of brain tubulin carboxypeptidase and different from those of pancreatic carboxypeptidase A. In comparison with cultures at confluence, those at low cell density contained small (if any) amounts of carboxypeptidase activity associated with microtubules. In addition, the enzyme was shown to be associated only with cold-labile microtubules. The tubulin carboxypeptidase/microtubule association was also demonstrated in Chinese hamster ovary, NIH 3T3 and PC12 cells. Interestingly, this association was not observed in cultured embryonic brain cells. Our results demonstrate that tubulin carboxypeptidase is indeed associated with microtubules in living cells. Furthermore, the findings that this association occurs with a subset of microtubules and that its magnitude depends on the degree of confluence of the cell culture indicate that it could be part of the mechanism that regulates the tyrosination state of microtubules.

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35

Hendriks, D., S. Scharpé, M. Sande, and M. P. Lommaert. "Characterisation of a Carboxypeptidase in Human Serum Distinct from Carboxypeptidase N." Clinical Chemistry and Laboratory Medicine 27, no. 5 (1989): 277–86. http://dx.doi.org/10.1515/cclm.1989.27.5.277.

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36

Matsumura, E., T. Sato, and N. Toyoda. "Isolation and characterization of a microbial Arg/Lys carboxypeptidase, carboxypeptidase F." Letters in Applied Microbiology 20, no. 3 (March 1995): 157–59. http://dx.doi.org/10.1111/j.1472-765x.1995.tb00415.x.

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Zhloba, A. A., T. F. Subbotina, D. S. Lupan, V. A. Bogova, and O. A. Kusheleva. "Arginine and lysine as products of basic carboxypeptidase activity associated with fibrinolysis." Biomeditsinskaya Khimiya 59, no. 5 (2013): 570–77. http://dx.doi.org/10.18097/pbmc20135905570.

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Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products in the conditions close to those in vivo . Plasma samples from 15 patients with arterial hypertension were investigated. The coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by clot turbidity assay. Fibrinolysis led to a large and significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated to the degree of increase of these amino acids: r =-0.733 и -0.761 for arginine and lysine, respectively (p<0.05). The rates of amino acids liberation during fibrinolysis demonstrate different pattern: arginine generation had two maximums: at the beginning of clot lysis and at his end, whereas the liberation of lysine occurred mainly at the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential aminoacids.

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38

van der Linden, M. P. G., L. de Haan, O. Dideberg, and W. Keck. "Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli." Biochemical Journal 303, no. 2 (October 15, 1994): 357–62. http://dx.doi.org/10.1042/bj3030357.

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Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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39

Blinkovsky, Alexander M., Tony Byun, Kimberly M. Brown, and Elizabeth J. Golightly. "Purification, Characterization, and Heterologous Expression in Fusarium venenatum of a Novel Serine Carboxypeptidase from Aspergillus oryzae." Applied and Environmental Microbiology 65, no. 8 (August 1, 1999): 3298–303. http://dx.doi.org/10.1128/aem.65.8.3298-3303.1999.

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ABSTRACT A novel serine carboxypeptidase (EC 3.4.16.1 ) was found in anAspergillus oryzae fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25°C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60°C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform aFusarium venenatum host strain. The transformed strain ofF. venenatum expressed an active recombinant carboxypeptidase. In F. venenatum, the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from A. oryzae. Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters.

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40

Jin, Kunlin, Steven H. Graham, Tetsuya Nagayama, Paul C. Goldsmith, David A. Greenberg, An Zhou, and Roger P. Simon. "Altered Expression of the Neuropeptide-Processing Enzyme Carboxypeptidase E in the Rat Brain after Global Ischemia." Journal of Cerebral Blood Flow & Metabolism 21, no. 12 (December 2001): 1422–29. http://dx.doi.org/10.1097/00004647-200112000-00006.

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Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia.

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41

Delk, A. S., P. R. Durie, T. S. Fletcher, and C. Largman. "Radioimmunoassay of active pancreatic enzymes in sera from patients with acute pancreatitis. I. Active carboxypeptidase B." Clinical Chemistry 31, no. 8 (August 1, 1985): 1294–300. http://dx.doi.org/10.1093/clinchem/31.8.1294.

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Abstract Previous studies have suggested that measurement of active enzymes in relation to proenzymes in serum of patients with pancreatitis may reflect the degree of zymogen activation in the gland. Here we describe the first single-tube assay for an active form of a pancreatic enzyme that is ordinarily synthesized as a proenzyme. Human procarboxypeptidase B, which we purified to near homogeneity, is approximately 13 000 Da larger than the active enzyme (EC 3.4.17.2). Antibodies specific for active carboxypeptidase B were obtained by affinity chromatography of anti-carboxypeptidase B antisera on a gel containing procarboxypeptidase B, then used to develop a single-tube radioimmunoassay for measuring active carboxypeptidase B in serum. Using this assay, we were able to detect, for the first time, active carboxypeptidase B in sera from patients with acute pancreatitis. Preliminary data show a correlation between the serum concentrations of active carboxypeptidase B and those of active trypsin complexed with serum inhibitors, but no correlation with serum amylase values.

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42

Harty, Derek W. S., and Neil Hunter. "Carboxypeptidase activity common to viridans group streptococci cleaves angiotensin I to angiotensin II: an activity homologous to angiotensin-converting enzyme (ACE)." Microbiology 157, no. 7 (July 1, 2011): 2143–51. http://dx.doi.org/10.1099/mic.0.048710-0.

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We have found that Streptococcus gordonii FSS2, an infective endocarditis (IE) isolate, expresses a dipeptidyl-carboxypeptidase with activity homologous to angiotensin-converting enzyme (ACE). The carboxypeptidase activity was purified to homogeneity as a complex/aggregate from a bacterial surface extract and was also active as a 165 kDa monomer. The specific activity for the carboxypeptidase activity was eightfold higher than that for recombinant human ACE. Selected ACE inhibitors, captopril, lisinopril and enalapril, did not inhibit the ACE activity. The carboxypeptidase also hydrolysed the Aα and Bβ-chains of human fibrinogen, which resulted in impaired fibrin formation by thrombin. The gene encoding ACE carboxypeptidase activity was sequenced and the inferred polypeptide product showed 99 % amino acid homology to SGO_0566, sgc, ‘challisin’ of S. gordonii CL1 Challis, and had no significant amino acid sequence homology to human ACE. Homologues of challisin ACE activity were commonly detected among the viridans group streptococci most often associated with IE.

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43

Guest, P. C., D. Pipeleers, J. Rossier, C. J. Rhodes, and J. C. Hutton. "Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans." Biochemical Journal 264, no. 2 (December 1, 1989): 503–8. http://dx.doi.org/10.1042/bj2640503.

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The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.

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44

Du, Xiao-Yan, Brian A. Zabel, Samantha J. Allen, Tracy M. Handel, Peter P. Lee, Eugene C. Butcher, and Lawrence L. Leung. "Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (TAFIa) and Platelets." Blood 110, no. 11 (November 16, 2007): 408. http://dx.doi.org/10.1182/blood.v110.11.408.408.

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Abstract Chemerin is a potent chemoattractant for cells that express the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. Chemerin circulates in the blood in a relatively inactive form (prochemerin). Its chemotactic activity is unleashed following proteolytic cleavage of carboxyl-terminal amino acids by serine proteases including plasmin, factor XIIa and VIIa. Recruitment of plasmacytoid dendritic cells and macrophages by chemerin may play a role in local tissue immune and inflammatory responses. The shortest bioactive chemerin peptide NH2-YFPGQFAFS-COOH (9mer) is present in the carboxyl-terminal domain. In this work, we show that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activatable fibrinolysis inhibitor (TAFIa)) (30 nM) remove the C-terminal lysine (K) from YFPGQFAFSK (10mer) (200nM) and enhance the migration of CMKLR1-transfected cells by ∼16-fold. To investigate if sequential proteolysis by plasmin and carboxypeptidases can modulate the activity of chemerin peptides, we generated the carboxyl-terminal 15mer of prochemerin, NH2-YFPGQFAFSKALPRS-COOH. Plasmin cleavage (1 μM) generated a 10mer, which was further processed to 9mer by CPN or CPB (30 nM) cleavage. These sequential cleavages were paralleled by corresponding increases in chemotactic activity. At concentrations as high as 1 μM the 15 mer did not induce chemotaxis; after plasmin cleavage and conversion to 10mer, however, significant chemotactic activity was demonstrated. Treatment with CPN or CPB further enhanced this chemotactic activity. We observed a similar enhancement in bioactivity following sequential plasmin/CPN-or-CPB cleavage of full-length prochemerin. Endogenous plasma CPN supports the activation (∼2.5 fold increase in bioactivity) of plasmin-cleaved prochemerin (0.2 nM). This activation was blocked by incubating plasma with MGTA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a specific CPN inhibitor. In addition, we show that platelets are a rich source of chemerin (20–30 ng of chemerin/5x108 platelets). Chemerin released from activated platelets triggers CMKLR1-transfectant chemotaxis, which was blocked by anti-chemerin antibodies. Thus, the circulating humoral factors reported here (platelets, serine proteases, and carboxypeptidases) may contribute to the regulation of chemerin bioactivity in vivo and therefore play a critical role in CMKLR1-mediated immune responses.

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45

Milkowski, C. "Serine carboxypeptidase-like acyltransferases." Phytochemistry 65, no. 5 (March 2004): 517–24. http://dx.doi.org/10.1016/j.phytochem.2003.12.018.

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46

Kalinina, Elena V., and Lloyd D. Fricker. "Palmitoylation of Carboxypeptidase D." Journal of Biological Chemistry 278, no. 11 (January 6, 2003): 9244–49. http://dx.doi.org/10.1074/jbc.m209379200.

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47

Hurtley, Stella M. "Tubulin carboxypeptidase identity revealed." Science 358, no. 6369 (December 14, 2017): 1397.18–1399. http://dx.doi.org/10.1126/science.358.6369.1397-r.

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48

Willemse, Johan L., David Chen, and Dirk F. Hendriks. "Major carboxypeptidase N deficiency." Clinica Chimica Acta 389, no. 1-2 (March 2008): 181–82. http://dx.doi.org/10.1016/j.cca.2007.11.008.

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49

Mock, William L., and Donghong Xu. "Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates." Bioorganic & Medicinal Chemistry Letters 9, no. 2 (January 1999): 187–92. http://dx.doi.org/10.1016/s0960-894x(98)00715-x.

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50

Campbell, William D., Eliada Lazoura, Noriko Okada, and Hidechika Okada. "Inactivation of C3a and C5a Octapeptides by Carboxypeptidase R and Carboxypeptidase N." Microbiology and Immunology 46, no. 2 (February 2002): 131–34. http://dx.doi.org/10.1111/j.1348-0421.2002.tb02669.x.

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Journal articles on the topic 'Carboxypeptidase A'

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