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 613 records · Page 34Linked to original sources

PubMed1989

Isolation and sequence analysis of cDNA for rat carboxypeptidase E [EC 3.4.17.10], a neuropeptide processing enzyme.

Carboxypeptidase E (CPE) is the carboxypeptidase B-like enzyme associated with the biosynthesis of numerous peptide hormones and neurotransmitters. This enzyme has been previously purified to homogeneity from bovine tissues, and cDNA clones (non-full length) isolated from a bovine pituitary cDNA library. In the present study, cDNA encoding full-length rat CPE has been isolated and sequenced. Both the nucleotide and amino acid sequences of rat CPE show substantial homology with the bovine sequences. The bovine and rat nucleotide sequences are homologous within the entire coding region, as well as within several portions of the 3'-untranslated region. The predicted amino acid sequence of rat CPE is greater than 90% homologous with the bovine enzyme. Northern blot analyses indicate a single species of CPE mRNA approximately 2100 nucleotides in length to be present in many neural and endocrine tissues. High levels of CPE mRNA are present in rat hypothalamus, hippocampus, midbrain, striatum, and cerebral cortex; and moderate levels are present in the brain stem, cerebellum, heart, adrenal, and eye. Low levels are detected in testis and duodenum, but not in liver or thymus. This tissue-specific expression of CPE mRNA is consistent with the proposed role for this enzyme in the production of numerous peptide hormones and neurotransmitters.

Adrenal Glands↗

PubMed1991

Structural characterization of the rat carboxypeptidase-E gene.

Several genomic clones encoding carboxypeptidase-E (CPE) have been isolated and partially sequenced. Southern blot analysis indicates that a single copy of this gene is present in the rat genome. The entire gene spans approximately 50 kilobases and consists of nine exons, each of which contains protein-coding regions. Only one of the exon/intron junctions of the rat CPE gene is present in a comparable position within the genes for carboxypeptidase-A and -B, both of which are only 17-21% homologous to CPE at the amino acid level. Nuclease protection analysis shows that alternative splicing of exons 7, 8, and 9 does not occur, indicating that the heterogeneity of the C-terminal region of CPE is due to posttranslational processing. Primer extension and nuclease protection analyses have identified the 5' end of CPE mRNA to be 105 nucleotides up-stream from the ATG used for protein translation. The 5' flanking region does not contain TATA and/or CCAAT boxes in the near vicinity of the transcription initiation site. The 5' flanking region is GC rich, containing 70% GC residues over nucleotides -1 to -150 (relative to the transcription initiation site). Putative consensus sites for the enhancer elements SP-1, NF-1, Pan-1, and AP-2 are present in the region from -60 to -330. Since this report describes the first neuropeptide-processing enzyme gene to be partially sequenced, it is not possible to compare the sequence with those of other processing enzymes that show similar tissue-specific expression. However, comparison of the CPE sequence with 5' flanking regions of other neuroendocrine genes has revealed a short region (12-18 nucleotides) that is highly conserved among CPE, neuropeptide-Y, oxytocin, insulin, and tyrosine hydroxylase genes.

Amino Acid Sequence↗

PubMed1999

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor.

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.

Animals↗

PubMed1998

Adult carboxypeptidase E-deficient fat/fat mice have a near-total depletion of brain CCK 8 accompanied by a massive accumulation of glycine and arginine extended CCK: identification of CCK 8 Gly as the immediate precursor of CCK 8 in rodent brain.

Cholecystokinin (CCK) amide concentrations were reduced over 85% in all the major brain regions of carboxypeptidase E (Cpe)(fat)/Cpe(fat) mice in comparison to control mice. Using an radioimmunoassay (RIA) specific for glycine-extended CCK (CCK Gly), low levels of CCK Gly were detected in control (0.65 ng/g tissue) and were even lower in Cpe(fat)/Cpe(fat) (0.246 ng/g) mice brain extracts. After treatment with carboxypeptidase B, the level of CCK Gly in Cpe(fat)/Cpe(fat) in these brain extracts was elevated to 33.5 ng/g, about 51-fold higher than in control. On gel-filtration chromatography and high-performance liquid chromatography (HPLC), this material coeluted with CCK 8 Gly. These results demonstrate that CPE is required for the correct processing of arginine- and glycine-extended CCK in all major regions of the mouse brain. These results support the hypothesis that CCK 8 Gly is the immediate precursor of CCK 8 amide in mouse brain, not larger amidated forms like CCK 22 or CCK 33.

Animals↗

PubMed1994

Redetermination and refinement of the complex of benzylsuccinic acid with thermolysin and its relation to the complex with carboxypeptidase A.

The complex of benzylsuccinic acid with thermolysin has been redetermined at 1.7-A resolution and refined to a crystallographic residual of 15.7%. In contrast to the prior study, which was to 2.3-A resolution, and without the benefit of refinement (Bolognesi, M. C. and Matthews, B. W. (1979) J. Biol. Chem. 254, 634-639), the present analysis shows that it is the D- rather than the L-isomer of benzylsuccinic acid that binds. The stereochemistry of the zinc-carboxylate interaction is now seen to be syn, as is also observed in all known zinc-carboxylate complexes of both thermolysin and carboxypeptidase A. The mode of binding of the beta-carboxylate resembles the presumed geometry of the tetrahedral transition state and, as such, is consistent with the commonly accepted mechanism of action of thermolysin and of carboxypeptidase A.

Binding Sites↗

PubMed1999

Enzymic characterization of a novel member of the regulatory B-like carboxypeptidase with transcriptional repression function: stimulation of enzymic activity by its target DNA.

The adipocyte-enhancer binding protein (AEBP) 1 is a novel transcriptional repressor with carboxypeptidase (CP) activity. AEBP1 binds to a regulatory sequence (termed adipocyte enhancer 1, AE-1) located in the proximal promoter region of the adipose P2 (aP2) gene, which encodes the adipocyte fatty-acid binding protein. Sequence comparisons and kinetic studies using known carboxypeptidase substrates, activators and inhibitors have characterized AEBP1 as a member of the regulatory B-like CP family. Significantly, the inherent CP activity of AEBP1 is stimulated by the AE-1 sequence. Our results indicate that AEBP1 is activated by a novel mechanism, wherby the direct binding of DNA enhances its protease activity. These results represent the first demonstration of DNA-mediated regulation of CP activity.

Binding Sites↗

PubMedDate not supplied

[Effect of sodium hydroxybutyrate on carboxypeptidase H activity and on angiotensin-converting enzyme in different regions of the rat brain].

It is revealed, that analogue of the gamma-aminobutyric acid--sodium hydroxybutyrate causes decrease of activities carboxypeptidase H and angiotensin converting enzyme in pituitary gland, hypothalamus and striatum. The most expressed changes of enzyme activities were observed in pituitary gland and hypothalamus. The activity of carboxypeptidase H changes more essentially, than one of angiotensin converting enzyme. The assumption one of mechanisms of influence the hydroxybutyric acid and, possible, the gamma-aminobutyric acid, on neuropeptides level is changes of activity of enzyme of biologically active peptides exchange is expressed.

Animals↗

PubMed2003

The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions.

Proneuropeptides are packaged into dense-core vesicles in which they are processed into active peptides by copackaged enzymes. Proprotein convertases (PCs) cleave precursors after dibasic residues, and carboxypeptidases remove basic residues from the C terminals. We show here that the Caenorhabditis elegans egl-21 gene encodes a protein that is very similar to carboxypeptidase E (CPE) and is broadly expressed in the nervous system. Mutants lacking either egl-21 CPE or egl-3, which encodes the C. elegans ortholog of PC type 2 (PC2), were defective for processing endogenously expressed FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides (FaRPs). Mutants lacking the unc-104 kinesin motor protein were defective for anterograde movement of dense-core vesicle components, including egl-3 PC2, egl-21 CPE, and FaRPs. We provide evidence that egl-3 PC2 and egl-21 CPE mutants have diminished acetylcholine release at neuromuscular junctions (NMJs). Taken together, these results suggest that egl-21 CPE and egl-3 PC2 process endogenous neuropeptides that facilitate acetylcholine release at C. elegans NMJs.

Acetylcholine↗

PubMedDate not supplied

[Effect of stress factors on carboxypeptidase H activity in areas of the rat brain].

It is established that carboxypeptidase H activity increases as affected by various stress factors in the rat brain departments. The increase of the enzyme activity because of the emotional-pain stress is of continuous character. Possible role of carboxypeptidase H in the development of the stress response is discussed.

Animals↗

PubMed2005

[The effect of ethanol consumption by dams on the offspring locomotion in the open field test and carboxypeptidase activities in the rat brain and adrenal medulla].

Consumption of dams ethanol increased the posterity locomotion activity in open field test. The increase in female rats was higher then in male ones. Differences in the carboxypeptidase H and PMSF-inhibited carboxypeptidase activities between the brain regions and adrenal medulla of prenatally exposed to ethanol and intact rats were found. The changing of enzyme activities in female rats was higher then in male ones. It is possible that dams ethanol consumption induced profound changes in locomotion mediated, at least partially, by changes in the rate of proteolytic processing of neuropeptide precursors.

Adrenal Glands↗

PubMed1992

Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.

Amino Acid Sequence↗

PubMed1991

Thermostabilization of carboxypeptidase A by interaction with its monoclonal antibodies.

The effect of interaction of carboxypeptidase A (CPA) with three monoclonal antibodies, each with a different epitope (CP 10, CP 9, and CP 8), on the heat stabilization of enzymes is described. These monoclonal antibodies bind to CPA with a relatively high binding constant (approximately 10(8) M-1) and do not affect its catalytic properties. Intact carboxypeptidase A lost more than 95 and 90% of its esterase and peptidase activities within 120 min at 50 degrees C. The monoclonal antibodies increased the thermal stability of the enzyme by 90 and 60%, as compared with the peptidase and esterase initial activities, respectively. Binding of these monoclonal antibodies, alone or in pairs, to the enzyme epitopes that are supposedly involved in heat denaturation of CPA result in stabilization of the conformation of the enzyme. The effect of thermostabilization by monoclonal antibodies was more pronounced with respect to peptidase activity than to esterase activity, indicating that these activities follow different reaction mechanisms. Since properly selected monoclonal antibodies can be prepared against virtually any enzyme, their immunocomplexation may provide a general and convenient method for stabilization of the enzyme conformation to heat denaturation, without affecting the catalytic properties.

Antibodies, Monoclonal↗

PubMed1991

Structural evolution of an enzyme specificity. The structure of rat carboxypeptidase A2 at 1.9-A resolution.

The structure of rat carboxypeptidase A2 (CPA2), which has a unique specificity for tryptophan-containing COOH-terminal peptides, has been determined in an unliganded state at 1.9-A resolution and refined to a crystallographic R-factor of 18.3%. Comparison of the structure of CPA2 with that of bovine carboxypeptidase A (referred to here as CPA1) reveals that the specificity of the former for larger amino acids probably arises from two amino acid replacements within the binding cavity (Thr268----Ala and Leu203----Met), coupled with differences in the positions of conserved residues in a surface loop on one face of the specificity pocket. The position of the reactive-site surface loop may be affected also by a disulfide bridge between Cys210 and Cys244. In this unliganded form of the enzyme, Tyr248 takes up a position interior to the specificity pocket and is distinct from that observed in bovine CPA1. The structural differences between CPA1 and CPA2 correlate strongly with crystallographically determined temperature factors and thus appear to be largest where the enzyme is flexible.

Amino Acid Sequence↗

PubMed1990

Two soluble forms of bovine carboxypeptidase H have different NH2-terminal sequences.

Carboxypeptidase H is an important enzyme in the biosynthesis of many peptide hormones. Development of a rapid isolation procedure led to the purification of two soluble forms from acidic extracts of bovine pituitary glands. These two forms differed in apparent molecular size (56 and 53 kDa). Both forms were found in the anterior lobe while only the 53-kDa form was found in posterior lobe. Digestion with N-glycosidase F demonstrated that these two forms are not due to alternative glycosylation of a common polypeptide core. Both forms bind antibodies raised against a COOH-terminal peptide of the full-length protein showing that the difference between them is not due to proteolysis at the COOH terminus. These results also argue against the idea that proteolysis of COOH-terminal domains converts the membrane-associated form of this protein into a soluble form. NH2-terminal sequence analysis demonstrated different NH2 termini. The NH2-terminal sequence of the 56-kDa form begins at the site predicted for signal peptide cleavage. Ion-exchange chromatography resolved the 56-kDa form from the 53-kDa form. The two forms were catalytically active with very similar properties. These results show that bovine carboxypeptidase H can be posttranslationally processed at alternative sites and provide evidence against the idea of a prosequence that must be removed before enzyme activity can be expressed.

Amino Acid Sequence↗

PubMed1990

An arginine carboxypeptidase generated during coagulation is diminished or absent in patients with rheumatoid arthritis.

A novel arginine carboxypeptidase that is generated during blood coagulation is diminished in sera obtained from patients with rheumatoid arthritis. The enzyme, which is unrelated to carboxypeptidase N, is more effective than lysine in removing terminal arginine from small synthetic substrates and may function in vivo in the removal of terminal arginine from inflammatory peptides such as C3a and C5a. Either diminished levels of this enzyme or an inability to generate it may be an important consideration in the mechanisms involved in the local inflammation that is observed in patients with rheumatoid arthritis.

Arthritis, Rheumatoid↗

PubMed1990

Carboxypeptidase E (enkephalin convertase): mRNA distribution in rat brain by in situ hybridization.

Carboxypeptidase E (CPE), also referred to as enkephalin convertase or carboxypeptidase H (EC 3.4.17.10), is present in neurotransmitter secretory granules and can remove C-terminal basic residues following endopeptidase cleavage during peptide processing. Using in situ hybridization with 35S-labeled oligonucleotide probes, we have mapped the localization of CPE mRNA in the rat brain. Specificity for CPE was confirmed by control experiments, which included production of identical patterns hybridization with 3 different antisense oligonucleotide probes, loss of label with RNase pretreatment of sections or co-incubation with excess unlabeled probe, and lack of labeling with sense orientation probes. In addition, the regional distribution of CPE mRNA by Northern blot analysis corresponded with distribution of labeling by in situ hybridization. The highest levels of CPE mRNA were found to be present in the pyramidal cells of the hippocampus, the pituitary anterior and intermediate lobes, the ependymal cells of the lateral ventricle, the endopiriform nucleus, the basolateral amygdala, the supraoptic nucleus, and the paraventricular nucleus. Intermediate levels were present in the thalamus, medial geniculate nucleus, lateral septal nucleus, piriform and entorhinal cortex, nucleus of the tractus solitarius, cerebellar cortex, pontine nuclei, and inferior olive. The lowest levels were found in the hippocampal granule cell layer, lateral hypothalamus, globus pallidus, and brain stem reticular formation. Ibotenic acid lesions of the hippocampus eliminated the majority of the label, which had been present over pyramidal cells, though labeling was increased over areas of reactive gliosis, suggesting that activated astrocytes can also synthesize CPE mRNA. In general, the localization of CPE mRNA in the rat brain corresponded to the distribution of enkephalin and other peptide neurotransmitter-synthesizing neurons, though CPE mRNA was also present in neurons that do not secrete known peptides and in reactive glia. The widespread yet specific localization of CPE mRNA in the rat brain suggests it may be an excellent marker for peptide synthesizing cells in the CNS.

Adrenal Glands↗

PubMed1986

Opioid agonist activity of ICI 174864 and its carboxypeptidase degradation product, LY281217.

The opioid peptide antagonist, ICI 174864 ([allyl]2-Tyr-alpha-amino-isobutyric acid (Aib)-Aib-Phe-Leu-OH), can produce analgesic effects in mice. The present study explored the possibility that ICI 174864 1) may have affinity and agonist efficacy at mu receptors and/or 2) may form a carboxypeptidase degradation product in vivo that possesses mu agonist activity. In vitro, ICI 174864 (10(-7) to 10(-4) M) inhibited the twitch in the electrically stimulated mouse vas deferens (ED50 = 90 microM) and guinea pig ileum (ED50 greater than 10(-4) M). The in vitro partial agonist activity of ICI 174864 was due to interaction with delta and not mu receptors because the apparent dissociation constant for naloxone using ICI 174864 as the agonist was similar to the apparent dissociation constant for the interaction of naloxone with delta and not mu receptors. Thus, ICI 174864 is a weak partial agonist at delta receptors with little affinity or efficacy at mu receptors. The incubation of ICI 174864 with carboxypeptidase A generated a peptide, LY281217 [(allyl)2-Tyr-Aib-Aib-Phe-OH], which was a more potent agonist in the mouse vas deferens and guinea pig ileum than ICI 174864. The agonist activity of LY281217 was due to interaction with mu and not delta receptors because LY281217 was approximately 100-fold more potent than ICI 174864 as an agonist in the guinea pig ileum, the apparent dissociation constant for naloxone using LY281217 as the agonist was similar to the apparent dissociation constant for the interaction of naloxone with mu receptors and the delta selective antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics, Opioid↗

PubMed1986

A novel method for the isolation of carboxypeptidase B.

Carboxypeptidase B was isolated from porcine pancreas by selective heat treatment of the autolyzed tissue at 60 degrees C and pH 6.0 the presence of phosphate ions. The heat treatment was followed by ammonium sulfate fractionation and ion-exchange in a batch system. The method could also be used for the isolation of carboxypeptidase B from beef pancreas.

Animals↗