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PubMed1987

Determination of carboxypeptidase A using N-acetyl-phenylalanyl-3-thiaphenylalanine as substrate: application to a direct serum assay.

N-Acetyl-L-phenylalanyl-L-3-thiaphenylalanine has been shown to be a substrate for carboxypeptidase A. Hydrolysis of the compound obeys Michaelis-Menten kinetics with a KM of 0.22 mM and a kcat of 6720 min-1 at 22 degrees C. A colorimetric assay, employing Ellman's reagent to detect the thiophenol released upon cleavage of the peptide, has been developed. The assay can be used for the direct determination of carboxypeptidase A in serum.

Animals↗

PubMed1978

Isoelectric focusing of carboxypeptidase N.

Carboxypeptidase N was partially purified on a TEAE-cellulose column and subjected to isoelectric focusing in sucrose gradient columns containing ampholine gradients of pH range 3-10 and 4-8. Activity separated into two major peaks with pI values of pH 3.8 and 4.3. Both peaks were totally converted to an active desialated enzyme with isoelectric point of pH 5.2 to 5.4. These results indicate that carboxypeptidase N is a sialoprotein with at least two forms, differing in sialic acid content, in serum. Catalytic activity is not dependent upon sialic acid but the latter may possibly influence stability since loss of activity occurred in the desialated enzyme with repeat focusing.

Carboxypeptidases↗

PubMed1979

The solubilisation of the membrane-bound D-alanyl-D-alanine carboxypeptidase of Bacillus coagulans NCIB 9365.

Protoplast membranes and the particulate D,D-carboxypeptidase of Bacillus coagulans NCIB 9365 were extremely resistant to disruption by either detergents or urea. A combination of urea and the non-ionic detergent Genapol X-100 was required to achieve a significant solubilisation of membrane protein and D,D-carboxypeptidase in an active form; the pH optimum for this treatment was pH 7.5. Solubilisation of the enzyme was accompanied by a two-fold enhancement of activity. Kinetic results indicated that the enhancement may be due to an alteration in the conformation of the enzyme following disruption of membrane structure.

Bacillus↗

PubMed1980

Modification of the carboxypeptidase A active site residue Glu-270 prevents interaction with a protein protease inhibitor from Ascaris.

The involvement of two active site residues of carboxypeptidase A in binding a protein inhibitor from Ascaris was studied. Glu-270 was modified with N-ethyl-5-phenylisoxazolium-3'-sulfonate and Tyr-248 was modified with tetranitromethane or diazotized arsanilic acid. Modification of Glu-270 abolished protein inhibitor binding and Glu-270 was protected from modification when the enzyme was bound to the protein inhibitor. In contrast, modification of Tyr-248 did not abolish protein inhibitor binding, nor did such binding protect Tyr-248 from modification. The absorption isosbestic point of arsanilazocarboxypeptidase A (Tyr-248 chemically modified) underwent a blue shift from 428 to 416 nm when the modified enzyme was bound to the protein inhibitor between pH 7.7 and 9.0. The 416 nm isosbestic point is characteristic of the loss of interaction between modified Tyr-248 and the active site zinc ion. These results with a protein inhibitor can be compared to substrate catalysis in which Tyr-248 moves away from the active site zinc ion of carboxypeptidase A when substrate binds. The close association of Glu-270 with Ascaris inhibitor interaction is consistent with other results which show that of the active site residues, only the modification of Glu-270 completely abolishes catalysis.

Animals↗

PubMed1989

Design and chemical synthesis of a 32 residues chimeric microprotein inhibiting both trypsin and carboxypeptidase A.

A chimeric peptide, 32 amino acids long, bearing two active sites, one inhibiting trypsin (Kd = 1.8 10(-9) M), one carboxypeptidase A (Kd = 3 10(-9) M), was designed and synthesized. It is a "squash inhibitor" (EETI II, 28 amino acids) elongated with the 4 amino acids from the C-terminus of the potato carboxypeptidase inhibitor. It has 3 disulfide bridges assembled in the particular knotted topology shared by the two inhibitors, by conotoxin omega, and possibly by E. coli enterotoxin ST1b.

Amino Acid Sequence↗

PubMed1992

Three-dimensional structural resemblance between leucine aminopeptidase and carboxypeptidase A revealed by graph-theoretical techniques.

Using 3-D searching techniques based on algorithms derived from graph theory we have established a striking structural similarity between the structure of bovine carboxypeptidase A and that of the C-terminal domain of bovine leucine aminopeptidase. There is no significant sequence homology between the aminopeptidases and the carboxypeptidases but the strong structural relationship detected in this complex fold suggests that there may be a very remote divergent evolutionary relationship between these two enzyme classes.

Animals↗

PubMed1994

Ostrich (Struthio camelus) carboxypeptidase A: purification, kinetic properties and characterization of the pancreatic enzyme.

1. Carboxypeptidase A beta and carboxypeptidase A tau-type from the pancreas of the ostrich were purified by water extraction of acetone powder, aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. 2. The final preparations were homogeneous when subjected to SDS-PAGE and PAGE. The M(r) values obtained from SDS-PAGE for CPA beta and CPA tau-type were 34,600 and 34,400, respectively. 3. The effects of inhibitors (1,10 phenanthroline and indole-3-acetic acid), pH and temperature on CPA activity were examined. Ki-values for CPI, PPA, D-phe, D-trp and aminobenzylsuccinic acid were determined. 4. Km, kcat and kcat/Km values were determined for hipp-phe, cbz-gly-phe, cbz-(gly)2-phe, cbz-gly-leu, cbz-(gly)2-leu and cbz-(gly)2-val. 5. N-terminal sequencing and amino acid analysis were performed for CPA beta and CPA tau-type.

Amino Acid Sequence↗

PubMed1984

Carboxypeptidase B-like activity for the processing of enkephalin precursors in the membrane component of bovine adrenomedullary chromaffin granules.

Trypsin and carboxypeptidase B-like (CPB-like) peptidases should be involved in processing proenkephalin to form the small biologically active enkephalins. A carboxypeptidase B-like activity from the soluble fraction of bovine adrenomedullary chromaffin granules which converts 125I-(Met)-enkephalin-Arg6 to 125I-(Met)enkephalin has previously been described and characterized (1,2). In this study, CPB-like activity in the membrane bound component of chromaffin granules is characterized and compared with that in the soluble fraction. Membrane and soluble CPB activities cleaved 125I-(Met)enkephalin-Arg6 or 125I-(Met)enkephalin-Lys6 to form 125I-(Met)enkephalin. Like the soluble enzyme, the CPB-like activity in the membrane component had a pH optimum of 6.0, was inhibited by thiol agents (PCMPSA, CuCl2) and metal ion chelators (EDTA, 1,10-phenanthroline), and was stimulated by Co++. The membrane CPB-like activity appeared to be an intrinsic membrane protein, since 80% of the activity remained with the membranes after washing with 1.0 M NaCl. Membrane and soluble CPB-like activities in chromaffin granules appear to be similar enzymes.

Adrenal Medulla↗

PubMed1984

Interaction of carboxypeptidase A with monoclonal antibodies.

Several mouse monoclonal antibodies to carboxypeptidase A (CPA) were prepared and purified, and their interaction with the enzyme was investigated. CPA is a well-characterized zinc-containing exopeptidase exhibiting peptidase as well as esterase activity. The antibodies obtained could be classified as follows: antibodies inhibiting mainly the peptidase activity of the enzyme, antibodies inhibiting mainly its esterase activity, antibodies affecting both activities, and antibodies which bind to the enzyme but have no marked effect on its catalytic properties. Binding constants of approximately 10(6) M-1 were obtained for most of the antibody-enzyme complexes tested. Additional information on the effect of the monoclonal antibodies on the active site of CPA was obtained by determining the change in the circular dichroism spectra of arsanilazotyrosine-248 carboxypeptidase A occurring as a result of the interaction of the enzyme with the antibodies studied. These findings suggest that CPA possesses at least three different specific antigenic sites, and that the active site of the enzyme for its peptidase activity differs from that for its esterase activity, though both sites seem to overlap to a considerable extent.

Animals↗

PubMed1988

13C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A.

Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.

Acetates↗

PubMed1985

Properties of carboxypeptidase A-treated chicken gizzard tropomyosin.

Chicken gizzard tropomyosin was digested with carboxypeptidase A at the weight ratios of enzyme to substrate 1:200 and 1:50. Removal of about 16 C-terminal amino acid residues per tropomyosin molecule, at lower enzyme concentration, caused reversion of the effect on skeletal actomyosin ATPase activity from activating to inhibiting without an influence on polymerizability and actin-binding ability. Removal of about 26 C-terminal amino acid residues per molecule, at higher enzyme concentration, resulted in loss of polymerizability and actin binding ability. Digestion of gizzard tropomyosin with carboxypeptidase A has no dramatic effect on its binding to troponin T. The results show that not only the existence of head-to-tail overlapping regions but also their length is important for the functional properties of chicken gizzard tropomyosin.

Actins↗

PubMed1991

Substrate specificity of solvent viscosity effects in carboxypeptidase A catalyzed peptide hydrolysis.

We have investigated the viscosity of carboxypeptidase A catalyzed Bz-Gly-Phe hydrolysis at pH 7.5 (Tris) and 0.5 mol.l-1 NaCl over the range 10-100 mp, varied by addition of glycerol or sucrose. In contrast to previous reports of strong viscosity effects on the corresponding Cbz-Ala-Ala-Ala hydrolysis, both the catalytic constant and the Michaelis constant are virtually independent of viscosity over the 10-fold range investigated. Furthermore, the CD spectra of carboxypeptidase A in the high-viscosity media point to no change in the alpha-helix and beta-sheet structure in these media. The data are compatible either with a compacter, more rigid enzyme-substrate structure or with a more prominent role of intramolecular nuclear reorganization compared to protein reorganization for Bz-Gly-Phe than for Cbz-Ala-Ala-Ala. These views can be given a preciser frame in terms of stochastic chemical rate theory.

Amino Acid Sequence↗

PubMed1991

Primary structure and tissue distribution of anglerfish carboxypeptidase H.

Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides. This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase. These processing events have been observed in a variety of species, from yeast to mammals. As part of an effort to characterize prohormone processing enzymes in the anglerfish, Lophius americanus, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH). Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage lambda gt11. The library was screened with a probe derived from the cDNA for rat CPH. A 2400 base pair AF cDNA clone was isolated. This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH. The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide. The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes. Significantly, all of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme. Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones.

Amino Acid Sequence↗

PubMed1988

Serum acetylcholinesterase possesses trypsin-like and carboxypeptidase B-like activity.

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the electric organ of eel possesses a protease activity resembling that of a neuropeptide processing enzyme. To examine whether any mammalian AChEs possess a similar protease activity, the enzyme was purified, 110,000-fold from foetal bovine serum. Purified serum AChE cleaved 2 synthetic peptide substrates in a manner resembling the combined actions of trypsin-like and carboxypeptidase B-like enzymes. A synthetic fragment of preproenkephalin A (residues 97-107) containing a complete methionine-enkephalin sequence was cleaved by serum AChE to yield free methionine-enkephalin. The carboxypeptidase action of AChE was weakly stimulated by the presence of 100 microM CoCl2 suggesting the requirement of a metal ion for complete activity. The results support the hypothesis that in many tissues AChE may act as a neuropeptide processing enzyme.

Acetylcholinesterase↗

PubMed1989

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells.

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.

Angiotensin I↗

PubMed1992

In situ hybridization: mRNA levels of secretogranin II, neuropeptides and carboxypeptidase H in brains of salt-loaded and Brattleboro rats.

In situ hybridization was used to study the mRNA levels for vasopressin, galanin, secretogranin II and carboxypeptidase H in salt-loaded and Brattleboro rats. These animals represent an in vivo model for the chronic stimulation of the hypothalamo-neurohypophyseal neurons. As shown by immunelectron microscopy secretogranin II is co-stored with vasopressin in these neurons. In salt-loaded rats the levels of mRNA for vasopressin, galanin and secretogranin II are increased in the paraventricular and supraoptic nuclei. Analogous changes were observed for Brattleboro rats with the exception of the vasopressin message which was decreased in these animals. The secretogranin II message was also increased in neurons which do not contain the vasopressin mRNA, i.e. in magnocellular neurons of the lateral hypothalamus and in the subfornical organ. Carboxypeptidase H message was also found in the paraventricular and supraoptic nuclei and in the subfornical organ; however, in both models the changes in mRNA in these nuclei were much lower than those observed for the secretory peptides or non-existent. We conclude that chronic stimulation of vasopressin neurons leads to a concomitant up-regulation of the biosynthesis of neuropeptides and secretogranin II. We suggest that the secretogranin II message might be a useful general marker for identifying chronically stimulated neurons.

Animals↗

PubMed2005

Inhibition of carboxypeptidase U (TAFIa) activity improves rt-PA induced thrombolysis in a dog model of coronary artery thrombosis.

The objective of this study was to test the hypothesis if thrombolysis induced by recombinant tissue-type plasminogen activator, (rt-PA) could be facilitated by inhibiting carboxypeptidase U (CPU, active Thrombin Activatable Fibrinolysis Inhibitor, TAFIa) activity. The efficacy of rt-PA alone, or in combination with the carboxypeptidase inhibitor MERGETPA, was compared in a dog model of coronary artery thrombosis. Twenty dogs were randomised in two groups, one received rt-PA, 1 mg kg(-1), as intravenous infusion over 20 min starting 30 min after thrombus formation, and the other group received rt-PA, 1 mg kg(-1), as group one with the addition of MERGEPTA 5 mg kg(-1) starting 25 min prior to coronary artery occlusion and followed by infusion of 5 mg kg(-1) h(-1) until the end of experiment. Efficacy was assessed by determination of time to lysis, duration of patency and blood flow during patency. Both groups had similar baseline characteristics with respect to haemodynamic parameters, i.e., heart rate, blood pressure and coronary artery blood flow. Coadministration of rt-PA and MERGETPA resulted in significant decrease in time to lysis (15+/-1.5 min vs. 20+/-1.7 min, p=0.03), increased patency time (87+/-16 min vs. 46+/-12 min, p=0.047) and increased coronary blood flow during patency (1131 mL h(-1) vs. 405 mL h(-1), p=0.015), compared to rt-PA alone. These results indicate that an inhibitor of CPU activity may have a beneficial effect in patients undergoing thrombolytic therapy by attaining shorter time to reperfusion and improved coronary patency.

3-Mercaptopropionic Acid↗

PubMed1987

Alterations of carboxypeptidases N activities in patients with thyroid dysfunction.

Serum carboxypeptidases N (EC. 3.4.17.3) were determined spectrophotometrically in 87 patients with disturbances of thyroid function and in 131 euthyroid individuals, including 33 women taking estrogens for contraception. Carboxypeptidases N (CN) can be subdivided into CN1 and CN2, according to variable substrate affinity. In addition, measurements of blood pressure and in vitro tests of thyroid function were performed. In euthyroid controls, CN1 was negatively correlated with age. No significant differences between CN1 and CN2 have been observed with regard to sex. In patients with hyperthyroidism, the mean values of both enzymes were elevated, but this tendency proved to be significant only for CN2. In hypothyroid patients CN1 and CN2 levels were normal. Elevations of CN1 and CN2 in the hyperthyroid state seem not to be related to underlying immunological processes but to the thyroid hormone excess itself. In euthyroid women taking estrogens for contraception CN2 was also elevated.

Arginine↗