SEARCH · PubMed Health

Results for “CARBOXYPEPTIDASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources

PubMed1992

Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-antitrypsin in carcinomas of the exocrine pancreas of the dog.

The immunocytochemical detection of amylase, carboxypeptidase A, alpha 1-antitrypsin, carcinoembryonic antigen (CEA) and keratin in normal canine pancreatic tissue and in carcinomas of the exocrine pancreas of the dog is described. In the normal pancreas, the acinar cells contain amylase, carboxypeptidase and alpha 1-antitrypsin. The pancreatic ducts react with the antikeratin antibody. Twelve out of 14 pancreatic exocrine carcinomas showed immunoreaction with antiamylase antibody, and 10 with anticarboxypeptidase antibody. Five neoplasms reacted with anti-CEA antibody and three with the anti-alpha 1-antitrypsin antibody. It was not possible to find any systematic difference in the immunocytochemical profiles of acinar, tubular and undifferentiated carcinomas. These results indicate that immunocytochemical marking of amylase and carboxypeptidase is of value in the diagnosis of pancreatic neoplasms in the dog, especially if metastasis is the only material available for study and the tumour does not show any diagnostic feature on routine light microscope preparations.

Adenocarcinoma↗

PubMedDate not supplied

Thioamide substrate probes of metal-substrate interactions in carboxypeptidase A catalysis.

Three thioamide peptides in which the oxygen atom of the scissile peptide bond is replaced by sulfur (denoted by (= S)) were synthesized and found to be good, convenient substrates for carboxypeptidase A. The thioamide bond absorbs strongly in the ultraviolet region, and enzymatic hydrolysis is monitored easily using a continuously recording spectrophotometric assay. The reaction follows Michaelis-Menten kinetics with kcat values of 68, 9.0, and 3.7 sec-1 and Km values of 0.83, 0.81, and 0.53 mM for Z-Glu-Phe(= S)-Phe, Z-Gly-Ala(= S)-Phe, and Z-Phe(= S)-Phe, respectively. Activities of the thioamides and their oxygen amide analogs were determined with a series of metal-substituted carboxypeptidases. The Cd(II), Mn(II), Co(II), and Ni(II) enzymes exhibit 30%-35%, 60%-85%, 150%-190%, and 40%-55% of the Zn(II) enzyme activity with the amide substrates; this compares with 240%-970%, 0%-15%, 340%-840%, and 30%-140% of the Zn(II) activity, respectively, with the thioamides. The activity of the Cu(II) and Hg(II) enzymes is less than 3% toward all substrates. Cadmium, a thiophilic metal, yields an enzyme which is exceedingly active with the thioamides; the kcat/Km values are 2.4-9.7-fold higher than with Zn(II) carboxypeptidase. In contrast, Mn(II), which has a relatively low affinity for sulfur, yields an enzyme with correspondingly low activity toward the thioamides. The results are consistent with a mechanism for peptide bond hydrolysis in which the metal atom interacts with the substrate carbonyl atom during catalysis.

Amides↗

PubMed1988

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A.

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.

Amino Acid Sequence↗

PubMed1988

The bovine pro-carboxypeptidase A-S6 ternary complex: a rare case of a secreted protein complex.

Up to now, a non-covalent ternary complex in which the pro-carboxypeptidase A (subunit I) is associated to two functionally different proteins (subunits II and III) has only been found in the pancreas of ruminant species. In the other species studied so far, the pro-carboxypeptidase A is secreted either as a monomer or as a binary association with a functionally different protein. Subunit I is the immediate precursor of carboxypeptidase A. Subunit II is a chymotrypsinogen of the C-type, involved, like subunit I, in the degradation of proteins and peptides. Although closely related to the pancreatic serine endopeptidases, subunit III appears to be devoid of any specific enzymatic activity. Information about the spatial organization of the subunits in the ternary complex has been deduced from the sequential dissociation of the complex. In contrast to the mechanism of activation of subunits I and II, which is independent of their aggregation state, the catalytic properties of the resulting enzymes are sensitive to their aggregation state. Moreover, the structural basis of inactivity of subunit III as well as the physiological role of the ternary complex are also discussed in this review.

Amino Acid Sequence↗

PubMed1992

Effects of nerve growth factor on the biosynthesis of chromogranin A and B, secretogranin II and carboxypeptidase H in rat PC12 cells.

We investigated the biosynthesis of various constituents (chromogranins A and B, secretogranin II, carboxypeptidase H and synaptin/synaptophysin) of large dense core and small vesicles in PC12 cells. These cells were treated for up to 18 days with nerve growth factor. Peptide levels were determined by quantitative immunoblotting, their mRNAs by Northern blotting. Nerve growth factor treatment changed the levels of the various peptides investigated and their mRNAs in three patterns. Peptide and mRNA levels for chromogranin A and chromogranin B were increased on day 1 and then declined. Synaptin/synaptophysin levels slightly decreased from day 1 onwards. On the other hand secretogranin II increased steadily up to 217% for peptide levels and 257% for mRNA levels. For carboxypeptidase H for which only the mRNA could be determined an analogous behaviour was seen. Its mRNA after 14 days of nerve growth factor treatment was 459% of controls. These results establish that the biosynthesis of the secretory proteins chromogranin A, chromogranin B and secretogranin II is regulated differentially during nerve growth factor treatment. We suggest that neuronal differentiation is accompanied by an increased biosynthesis of secretogranin II. For carboxypeptidase H, the marked increase in mRNA levels after nerve growth factor treatment is the first example that the biosynthesis of this peptide is significantly up-regulated. Synaptin/synaptophysin biosynthesis is not increased although this peptide is a major constituent of small vesicles which increase in number during nerve growth factor treatment.

Animals↗

PubMed1995

Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity.

A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.

Amino Acid Sequence↗

PubMed1999

Determination of the activity of carboxypeptidase A in the blood of healthy human adults.

A sensitive and highly specific assay, utilizing N-acetyl-Phe-3-thiaPhe as substrate, has been designed to make possible the direct determination of carboxypeptidase A in human serum or plasma. Measurement of the enzyme's activity in the serum of 108 blood donors has established the basal concentration for healthy human adults to be 0.068+/-0.028 U/l (x+/-1 S.D.). This is equivalent to 0.34 microg/l of carboxypeptidase A. Such an extremely low baseline provides for a substantial dynamic range over which to assess pancreatic pathology. Previous claims in the literature for an 11 fold higher baseline have to be reexamined in view of the failure of the investigators to take into account the ability of the proenzyme form of carboxypeptidase A, which does occur in serum, to attack their substrates.

Adult↗

PubMed1989

Kinetic analysis of the carboxypeptidase A hydrolysis of oligopeptides by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic (HPLC)-based method was developed to follow the time course of the hydrolytic action of pancreatic carboxypeptidase A on oligopeptide substrates of the general formula benzoyl(glycyl)n-L-Phe, n being in the range 1-5. The proposed procedure is sensitive at the nanomolar level of substrate, but also allows the kinetics of substrate hydrolysis to be investigated over a wide range of concentrations. The prior quenching of the carboxypeptidase activity and the use of isocratic conditions for the rapid separation of the substrates and their products (in less than 8 min for the slowest one), allow the automation of the procedure, which could become an easy and versatile alternative to the commonly used spectrophotometric methods. A comparative evaluation of the use of a spectrophotometric method to measure carboxypeptidase A activity on the same substrates indicated that the latter is not valid for long oligopeptides (n greater than or equal to 2) at concentrations higher than 5 mM owing to an interference of a physico-chemical nature. For benzoylglycyl-L-Phe (n = 1), the apparent kinetic parameters were calculated by means of the HPLC method and by a well established spectrophotometric procedure, and both yielded similar values.

Animals↗

PubMed1998

Cleavage of atrial natriuretic peptide by a kidney membrane-bound carboxypeptidase A.

An enzymatic activity that cleaved the C-terminal Tyr of ANP (1-28) was characterized in human kidney microvillar membranes by using 125I-labeled rat ANP as substrate. This activity was inhibited by potato carboxypeptidase inhibitor (PCI) and 1.10 phenanthroline, suggesting that it corresponded to a metallo-carboxypeptidase. Solubilization experiments indicated that the carboxypeptidase activity could be recovered in the supernatant after 1% Triton X-100 extraction. As separation by ion exchange chromatography revealed several peaks of enzyme activity, PCI coupled to Sepharose was used for purification. This step resulted in a single protein band at 30 kDa, as analyzed by SDS-PAGE.

Atrial Natriuretic Factor↗

PubMed2000

Crystal structure of carboxypeptidase A complexed with D-cysteine at 1.75 A - inhibitor-induced conformational changes.

D-Cysteine differs from the antiarthritis drug D-penicillamine by only two methyl groups on the beta-carbon yet inhibits carboxypeptidase A (CPD) by a distinct mechanism: D-cysteine binds tightly to the active site zinc, while D-penicillamine catalyzes metal removal. To investigate the structural basis for this difference, we solved the crystal structure of carboxypeptidase A complexed with D-cysteine (D-Cys) at 1.75-A resolution. D-Cys binds the active site zinc with a sulfur ligand and forms additional interactions with surrounding side chains of the enzyme. The structure explains the difference in potency between D-Cys and L-Cys and provides insight into the mechanism of D-penicillamine inhibition. D-Cys binding induces a concerted motion of the side chains around the zinc ion, similar to that found in other carboxypeptidase-inhibitor crystal structures and along a limited path. Analysis of concerted motions of CPD and CPD-inhibitor crystal structures reveals a clustering of these structures into distinct groups. Using the restricted conformational flexibility of a drug target in this type of analysis could greatly enhance efficiency in drug design.

Binding Sites↗

PubMed1992

X-ray absorption fine structure study of the active site of zinc and cobalt carboxypeptidase A in their solution and crystalline forms.

A comparative study on the metal environment of Zn(II)-carboxypeptidase A (ZnCPD) and Co(II)-carboxypeptidase A (CoCPD) in their solution and crystalline forms using the X-ray absorption fine structure (XAFS) technique has been conducted. The first coordination sphere of Zn for ZnCPD in its solution state is found to consist of two distributions of atoms, with four atoms (N or O) located at an average distance of 2.03 +/- 0.01 A and one atom (N or O) located at 2.57 +/- 0.04 A. The four-atom distribution remains the same for ZnCPD in its crystalline state, but the fifth atom is found at 2.36 +/- 0.04 A. Examination of the higher coordination shell, between 2.7 and 4.2 A, reveals the presence of two imidazoles. Combined with X-ray crystallographic results, a structural model is proposed. The four atoms at an average distance of 2.03 A are assigned to the two delta 1 nitrogens of His-69 and His-196, one epsilon 1 oxygen of Glu-72, and the oxygen of a coordinated water molecule. The atom at 2.57 A for ZnCPD in solution is assigned to the epsilon 2 oxygen of Glu-72. The results for CoCPD in solution are similar with the four atoms at an average distance of 2.08 +/- 0.01 A and one atom at 2.50 +/- 0.04 A, which moves to 2.34 +/- 0.04 A in the crystalline enzyme. The intensity of the 3d "pip" peak for CoCPD is consistent with a distorted tetragonal metal geometry for the solution form of the enzyme which is converted to a more pentacoordinated metal site for the crystalline enzyme. The first shell distribution of crystalline CoCPD is quite disordered, which may be largely due to the disorder of His-69 and His-196 as indicated by higher shell analysis. Thus, the XAFS studies show that the metal coordination spheres in the zinc and cobalt enzymes are quite similar in the solution state but differ from their crystalline counterparts. The XAFS studies provide the necessary background for measurement of substrate- and inhibitor-promoted structural changes in the metal coordination sphere of the zinc and other metal-substituted carboxypeptidases in the solution state.

Animals↗

PubMed1990

Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro.

Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5' and 3' untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

Amino Acid Sequence↗

PubMed2000

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti.

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.

Aedes↗

PubMed1982

Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules.

A specific carboxypeptidase that converts enkephalin precursors into enkephalin in adrenal chromaffin granules has been purified and characterized. In the adrenal this enzyme, designated enkephalin convertase, is uniquely localized to the chromaffin granules, which contain enkephalin and precursor peptides. Enkephalin convertase is markedly stimulated by CoCl2 and inhibited by EDTA or 1,10-phenanthroline, unlike the lysosomal carboxypeptidase. The purified enzyme has a high affinity for the hexapeptides [Met5]- and [Leu5]enkephalin-Arg6 (51 and 83 microM, respectively) and a somewhat lower affinity for the hexapeptides [Met5]- and [Leu5]enkephalin-Lys6 (195 and 174 microM). Brain enkephalin convertase shows 10-fold regional variations, unlike other carboxypeptidases, which are uniformly distributed. Enkephalin convertase appears to be associated selectively and physiologically with biosynthesis of the enkephalins.

Adrenal Medulla↗

PubMed1997

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in cpefat mice associated with a carboxypeptidase E mutation.

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia-diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Adrenocorticotropic Hormone↗

PubMed1997

On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B.

The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.

Antifibrinolytic Agents↗

PubMed2001

The VanY(D) DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein.

VanD-type Enterococcus faecium BM4339 is constitutively resistant to vancomycin and to low levels of teicoplanin. This strain produces peptidoglycan precursors terminating in D-lactate but, unlike VanA- and VanB-type strains, E. faecium BM4339 has a mutated ddl ligase gene and cannot synthesize D-Ala-D-Ala. Consequently, although it possesses vanX(D) and vanY(D) genes, it should not require an active VanX-type DD-dipeptidase or a VanY-type DD-carboxypeptidase for resistance. The vanY(D) gene contains the signatures of a penicillin-binding protein (PBP) and is believed to encode a penicillin-sensitive DD-carboxypeptidase. The enzyme activity was found to be membrane-bound and inhibited by low concentrations of benzylpenicillin in membrane preparations and in intact bacteria, indicating that the active site was present on the outside surface of the membrane. The 38 kDa protein was revealed as a PBP present in more copies per cell than conventional PBPs and all the protein was accessible to benzylpenicillin added externally, confirming the localization of the active site. A glycopeptide-susceptible strain of E. faecium lacked this PBP, and the membrane-bound DD-carboxypeptidase activity was less than 5% of that of E. faecium BM4339. Although the active site of VanY(D) was external to the membrane, UDP-MurNAc-tetrapeptide was produced internally, probably from UDP-MurNAc-pentadepsipeptide. The presence of benzylpenicillin at low concentrations in the growth medium substantially reduced the amount of tetrapeptide produced, indicating that inhibition of VanY(D) by benzylpenicillin influenced production of peptidoglycan precursors internally. A model to explain these contrasting observations is proposed.

Bacterial Proteins↗

PubMed1982

Mechanistic implications of cyanide binding to carboxypeptidase B.

The putative metal coordinating ligand cyanide was used to study the effects of modifications of the metal coordination sphere on the spectral properties and catalytic activity of cobalt and zinc carboxypeptidases. The absorption spectra of Co2+-carboxypeptidase B in the presence of cyanide pointed to a direct interaction of the ligands with the metal. Gel-filtration experiments showed that the binding of one mole of ligand per mole of enzyme metal ion resulted in maximal spectral effects. Binding of cyanide to the metal ion as measured by absorption spectroscopy was inhibited by acetyl-L-arginine, a peptide pseudosubstrate, and by acetyl-D-arginine, a competitive peptide inhibitor. Addition of acetyl arginine to the enzyme-cyanide complex caused displacement of the ligand, as evidenced by the spectral parameters. Cyanide inhibited peptide hydrolysis in a partially noncompetitive manner, i.e. it did not prevent binding of the substrate to the enzyme but the enzyme-substrate-cyanide complex was hydrolyzed at a slower rate than the enzyme-substrate complex. The dissociation constant evaluated from kinetic studies for the binding of cyanide to Co2+-carboxypeptidase B was in good agreement with that obtained from spectral measurements. Hydrolysis of the ester analog of the basis peptide substrate was not affected by cyanide. Based on these data a model is proposed in which the peptide carboxyl group displaces the water molecule from the metal coordination sphere during catalysis without increasing the coordination number.

Animals↗