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 703 records · Page 39Linked to original sources

PubMed1985

Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys.

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.

Amino Acids↗

PubMed1989

A carboxypeptidase Y pulse method to study the accessibility of the C-terminal end during the refolding of ribonuclease A.

Carboxypeptidase Y pulses, applied after various times of refolding, were employed to probe the accessibility of the C-terminus of RNAase A during the refolding process. The increase in resistance against proteolytic cleavage was measured by determination of the amount of liberated C-terminal amino acids and by activity assays. The results indicate that the C-terminus of RNAase becomes inaccessible early in the course of refolding, if folding is carried out at low temperatures under conditions that effectively stabilize the native state. At higher temperatures (25 degrees C) or under conditions of marginal stability, intermediates are not populated and protection against proteolytic cleavage is not detectable before the formation of the native state. The method described may be used to monitor the accessibility of the C-terminus of various proteins during refolding. However, intermediates on the folding pathway can only be observed if the native state is stable against carboxypeptidase attack.

Carboxypeptidases↗

PubMed2002

Molecular characterization of three gut genes from Glossina morsitans morsitans: cathepsin B, zinc-metalloprotease and zinc-carboxypeptidase.

Insect gut enzymes are involved in digestion of dietary proteins. Additionally, these enzymes have been implicated in the process of pathogen establishment in several insects including the tsetse fly (Diptera:Glossinidae), which is the vector for African trypanosomes. Both the male and female tsetse can transmit trypanosomes and are strict blood feeders during all stages of their development. Here, we describe the molecular characterization of three gut genes: cathepsin B (GmCatB), zinc-metalloprotease (GmZmp) and zinc-carboxypeptidase (GmZcp). The cDNA for GmCatB encodes a protein for 340 amino acids with a predicted molecular mass of 38.2 kDa, while the 854 bp GmZmp cDNA encodes a protein of 254 amino acids with a molecular mass of 29 kDa. The GmZcp cDNA is 1319 bp in length and has a 354 amino acids open reading frame for coding a 40 kDa protein. All three cDNAs have signal peptide sequences associated with their N-terminal domains and structure analysis indicates that GmCatB and GmZmp are expressed as zymogens with pro-domains proteolytically removed for activity. The activation domain associated with the carboxypeptidase sequences is lacking in GmZcp. While GmCatB transcription is constitutive, teneral flies express very low levels of transcripts for GmZmp and GmZcp prior to the first bloodmeal. Transcription of all genes is induced and remains high throughout the digestion cycle within a few hours following the first bloodmeal ingestion. Both GmCatB and GmZcp are parasite responsive, with the expression of both genes being higher in trypanosome infected flies.

Amino Acid Sequence↗

PubMed2003

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.

Amino Acid Sequence↗

PubMed1998

Detection of carboxypeptidases as taxonomic markers for gram-negative bacteria.

The use of seven benzoyl-L-amino acids for the detection of carboxypeptidase activities in strains of Gram-negative aerobic and facultatively anaerobic bacteria is reported. A simple overnight assay was designed using ninhydrin for the demonstration of the amino acid released by hydrolysis. Detection of carboxypeptidase activity was shown to have some taxonomic relevance within the Enterobacteriaceae; it was also useful for differentiation within the genus Acinetobacter and for distinction between Pseudomonas aeruginosa and Pseudomonas fluorescens.

Amino Acids↗

PubMed1971

Inactivation of D-alanine carboxypeptidase by penicillins and cephalosporins is not lethal in Bacillus subtilis.

The D-alanine carboxypeptidase of Bacillus subtilis is a particulate enzyme that is irreversibly inactivated by penicillins and cephalosporins. However, the lethal concentrations of these antibiotics are not the same as those that inhibit enzymatic activity in vitro. 6-Aminopenicillanic acid inactivates at least 95% of the enzyme at nonlethal concentrations. Conversely, cephalothin is lethal at concentrations that do not inactivate the enzyme. Experiments with intact, growing cells confirm the results obtained in vitro. Therefore, a killing site distinct from the carboxypeptidase must be postulated.

Alanine↗

PubMed1972

Transpeptidase activity of Streptomyces D-alanyl-D carboxypeptidases.

In the presence of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala as donor, and either D-[(14)C]alanine, [(14)C]-glycine, or meso-[(3)H]diaminopimelic acid as acceptor, the DD carboxypeptidases from Streptomyces R61 and R39 catalyze a transpeptidation reaction with the release of terminal D-alanine from the donor and the formation of either N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-[(14)C]Ala, N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-[(14)C] Gly, or N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-meso- [(3)H]diaminopimelic acid. The reaction appears to be a true transpeptidation, and is not simply a "reversal of hydrolysis". Transpeptidation is inhibited by pencillin at concentrations that inhibit hydrolysis (carboxypeptidase action) of the donor peptide. There are differences in the specificity profiles of the Streptomyces enzymes for acceptor molecules:only the R61 enzyme used [(14)C]Gly-Gly as acceptor; transfer of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala to this acceptor resulted in the formation of N(alpha),N(epsilon)-diacetyl-Lys-D-Ala-[(14)C] Gly-Gly, with the synthesis of a (D-Ala-Gly) peptide bond in an endoposition.

Alanine↗

PubMed1973

Relation of detergent HLB number to solubilization and stabilization of D-alanine carboxypeptidase from Bacillus subtilis membranes.

The ability of various nonionic detergents to solubilize D-alanine carboxypeptidase and other membrane-bound enzymes was correlated to a physical property of the detergent, the HLB number. Only a fairly narrow range of detergents were effective solubilizing agents. Purified carboxypeptidase required either a detergent or detergent plus a lipid fraction for stability. Only those detergents effective in solubilizing the enzyme were effective in stabilizing it.

Alanine↗

PubMed1973

Conformations of arsanilazotyrosine-248 carboxypeptidase A alpha, beta, gamma, comparison of crystals and solution.

The spectra of the alpha, beta, and gamma forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, lambda(max) 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the absorption band at 510 nm is due to a complex between arsanilazotyrosine-248 and the active-site zinc atom. Two pK(app) values, 7.7 and 9.5, characterize the formation and dissociation of this arsanilazotyrosine-248.Zn complex. On titrations of the apoenzyme, the absorption band at 510 nm is completely absent at all pH values. Instead, there is a single pK(app), 9.4, due to the ionization of the azophenol, lambda(max) 485 nm. Substitution of other metals for zinc results in analogous intramolecular coordination complexes with absorption maxima and circular dichroism extrema characteristic of the particular metal. Similar data and conclusions have been derived from studies of heterocyclic azophenol.metal complexes. The present studies demonstrate that the conformation of the crystals of all generally available alpha, beta, and gamma forms of the arsanilazoenzyme differs from that of their solutions. The spectra of the modified x-ray crystals, however, differ from those of all other carboxypeptidase forms and crystal habits studied. The internal consistency of their data, their interpretation, and the conclusions of Lipscomb and coworkers [Proc. Nat. Acad. Sci. USA (1972) 69, 2850-2854] are examined. Dissimilar chemical modification or conformation is thought to underlie these differences. The arsanilazotyrosine-248.zinc complex is a sensitive, dynamic probe of environmental conditions. Its response to changes in pH and physical state of the enzyme suggest different orientation of the arsanilazotyrosine-248 side chain in solution from that in the crystal. This finding calls for reexamination of the basis of the substrate-induced conformation change which has been thought to be critical to the mechanism, postulated on the basis of the x-ray structure analysis performed at pH 7.5.

Arsenicals↗

PubMed1973

The role of a tyrosyl residue in the mechanism of action of carboxypeptidase B: luminescence studies.

The luminescence spectra of carboxypeptidase B indicate specific differences between the zinc and apoenzyme due to the state of tyrosyl residues presumably at the active site. These differences disappear when enzyme-substrate or enzyme-inhibitor complexes are formed, suggesting that they may reflect the interaction of a tyrosyl residue in the native enzyme with the catalytically essential zinc atom. An interpretation of the role of that tyrosyl residue in the mechanism of action of carboxypeptidase B is presented.

Animals↗

PubMed1975

Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of multiple conformational states in solution.

The red azoTyr-248-Zn complex of arnilazocarboxypeptidase, previously used to demonstrate differences in conformation of the enzyme in crystals and in solution, has now provided means to detect multiple conformations of the enzyme in solution by stopped-flow pH and temperature jump experiments. These studies identify two distinct processes. Er + H+ in equilibrium Ey (I), is the extremely rapid, Kfast about 10(5) sec-1, pH dependent dissociation of the metal complex. Ey in equilibrium Ey' (II), is much slower, Kslow about 5 sec-1, pH independent interconversion of two distinct populations of protein molecules, Ey and Ey', in which the yellow azo-Tyr-248 is different conformations. These two conformations can be differentiated readily by stopped-flow pH-jump experiments, since I is three to four orders of magnitude faster than II. Mathematical expressions derived from this mechanism accurately predict all observations over the pH range from 6.0 to 8.5. In a previous stopped-flow pH-jump experiment, Lipcomb and coworkers [Quiocho, F. A., McMurray, C. H. & Lipcomb, W. H. (1972), Proc. Nat. Acad. Sci. USA 69, 2850-2854] recognized only a single process with a rate constant of about 6 sec-1, but not the major, very rapid rate observed here. The failure to detect this fast process led to the postulation of a number of explanations intended to account for the detection of only a single, slow rate. The present observations show that the premise for those conjectures is not valid. The azoprobe reveals the existence of rapidly interconvertible substructures of carboxypeptidase A, and the results support the view that in solution, enzymes can adopt multiple, readily interconvertible and related conformations which could then either facilitate or impede catalysis. In crystals, rearrangement of molecular structure could be severely impaired or restricted, and crystallization might single out either active or inactive conformations. In the latter case, such crystals would have greatly reduced activities and markedly altered catalytic behavior, as is observed for carboxypeptidase A. In combination with detailed kinetic analysis of crystals, conformational analysis in solution should be a valuable guide to discern enzyme mechanisms and select crystals for x-ray structure analysis.

Azo Compounds↗

PubMed1977

Mutants of Escherichia coli lacking in highly penicillin-sensitive D-alanine carboxypeptidase activity.

Mutants of Escherichia coli lacking in the highly penicillin-sensitive enzyme activities of D-carboxy-peptidase, transpeptidase, and endopeptidase, and with the concomitant absence of penicillin-binding protein 4 of B.G. Spratt and A.B. Pardee [(1975) Nature 254, 516-517] were isolated. The defect of these mutants is ascribed to the lack of an enzyme, D-alanine carboxypeptidase Ib. Genetic mapping studies show the mutation (dacB) to be located at 68 min on the E. coli chromosome map. The dacB mutation results in the simultaneous loss of D-alanine carboxypeptidase and penicillin-binding protein 4. The mutants grew normally under a wide range of growth conditions. We conclude that the enzyme is not a necessary component for normal peptidoglycan biosynthesis in E. coli.

Alanine↗

PubMed1980

Structure and function of carboxypeptidase A alpha in supercooled water.

The spectral and enzymatic characteristics of chromophoric derivatives of carboxypeptidase A alpha (EC 3.4.17.1) have been examined at subzero temperatures in supercooled water-in-oil emulsions. Substrate and temperature dependencies of enzyme kinetics indicated the existence of a solution-like enzyme phase that greatly extends the temperature range (greater than 60 degrees C) over which the activity of this enzyme can be measured. The emulsion spectra were virtually identical to those of solutions over a wide range of temperatures. Subzero temperatures (less than -10 degrees C) may induce changes of enzyme conformation but not of geometry at the site of the metal atom, nor do they adversely affect activity at any of the temperatures studied. Both structure and function of carboxypeptidase A alpha can be examined in supercooled water under identical reaction conditions.

Carboxypeptidases↗

PubMed1991

Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity.

The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium album. We used this method of activation as a tool for the investigation of refolding procarboxypeptidase Y in vitro. The proenzyme, denatured in 6 M guanidinium chloride, is renatured efficiently after dilution of the denaturant, whereas the mature enzyme regains little activity in the same procedure. Changes in intrinsic fluorescence reveal the mature enzyme to be considerably more stable than the proenzyme toward denaturation with guanidinium chloride. This suggests that the propeptide induces a metastable structure important for overcoming energy barriers that might otherwise obstruct a productive folding pathway. The relatively large number of charged amino acid residues and a high theoretical potential for alpha-helix formation in the carboxypeptidase Y propeptide suggest a structural similarity to a number of other propeptides and heat shock proteins.

Carboxypeptidases↗

PubMed1995

Gut-specific transcriptional regulatory elements of the carboxypeptidase gene are conserved between black flies and Drosophila.

Millions of people die every year in the tropical world from diseases transmitted by hematophagous insects. Failure of conventional containment measures emphasizes the need for additional approaches, such as transformation of vector insects with genes that restrict vectorial capacity. The availability of an efficient promoter to drive foreign genes in transgenic insects is a necessary tool to test the feasibility of such approach. Here we characterize the putative promoter region of a black fly midgut carboxypeptidase gene and show that these sequences correctly direct the expression of a beta-glucuronidase reporter in Drosophila melanogaster. By histochemical staining and mRNA analysis, we found that the gene is expressed strongly and gut-specifically in the transgenic Drosophila. This gut-specific black fly carboxypeptidase promoter provides a valuable tool for the study of disease vectors.

Amino Acid Sequence↗

PubMed1995

Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor.

The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.

Amino Acid Sequence↗

PubMed2001

Cloning of a novel retinoid-inducible serine carboxypeptidase from vascular smooth muscle cells.

Retinoids block smooth muscle cell (SMC) proliferation and attenuate neointimal formation after vascular injury, presumably through retinoid receptor-mediated changes in gene expression. To identify target genes in SMC whose encoded proteins could contribute to such favorable biological effects, we performed a subtractive screen for retinoid-inducible genes in cultured SMC. Here, we report on the cloning and initial characterization of a novel retinoid-inducible serine carboxypeptidase (RISC). Expression of RISC is low in cultured SMC but progressively increases over a 5-day time-course treatment with all-trans-retinoic acid. A near full-length rat RISC cDNA was cloned and found to have a 452-amino acid open reading frame containing an amino-terminal signal sequence, followed by several conserved domains comprising the catalytic triad common to members of the serine carboxypeptidase family. In vitro transcription and translation experiments showed that the rat RISC cDNA generates an approximately 51-kDa protein. Confocal immunofluorescence microscopy of COS-7 cells transiently transfected with a RISC-His tag plasmid revealed cytosolic localization of the fusion protein. Western blotting studies using conditioned medium from transfected COS-7 cells suggest that RISC is a secreted protein. Tissue Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues analyzed; high level RISC expression was also observed in human kidney. In situ hybridization verified the localization of RISC to medial SMC of the adult rat aorta. Interestingly, expression in kidney was restricted to proximal convoluted tubules; little or no expression was observed in glomerular cells, distal convoluted and collecting tubules, or medullary cells. Radiation hybrid mapping studies placed the rat RISC locus on chromosome 10q. These studies reveal a novel retinoid-inducible protease whose activity may be involved in vascular wall and kidney homeostasis.

Amino Acid Sequence↗

PubMed2003

The multiple site binding of carboxypeptidase Y inhibitor (IC) to the cognate proteinase. Implications for the biological roles of the phosphatidylethanolamine-binding protein.

The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs). In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated. The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site. IC is able to form the complex with diisopropyl fluorophosphate-modified CPY. Tryptic digestion of the complex indicates that two fragments from IC are involved in complex formation with CPY. These findings demonstrate the multiple site binding of IC with CPY. Considering the fact that mouse PEBP has recently been identified as a novel thrombin inhibitor, the binding that characterizes the CPY-IC complex could be a common feature of PEBPs.

Amino Acid Sequence↗