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PubMed1978

Carboxypeptidase A activity of cultured skin fibroblasts and relationship to cystic fibrosis.

Properties of carboxypeptidase A of cultured skin fibroblasts from control and cystic fibrosis patients were studied using alpha-N-carbobenzoxy-L-glutamyl-L-tyrosine as substrate. Carboxypeptidase A was inhibited by thiomersal, cyanide, iodoacetate and N-ethylmaleimide in a similar manner for control and cystic fibrosis fibroblasts. Both trypsin and dithiothreitol treatment activated the enzyme, but 1,10-phenanthroline inhibited only in the presence of dithiothreitol. Both Zn2+ and Co2+ reversed this inhibition. Trypsin treatment of carboxypeptidase A produced a form of the enzyme having a higher KM value for both control and cystic fibrosis fibroblasts. Dithiothreitol treatment of control fibroblasts resulted in a form with similar properties to the trypsin activated form, but cystic fibrosis fibroblasts yielded a variant form with even higher KM and Vmax values. Since other properties were similar, it seems likely that this difference reflected binding of a molecule to the enzyme rather than of a defect in the enzyme.

Carboxypeptidases↗

PubMed1990

Carboxypeptidase activity in human urine from healthy subjects and renal disease patients.

We have attempted to estimate the carboxypeptidase activity in unconcentrated human urine by means of a colorimetric method in which Bz-Gly-Lys is employed as a substrate. Upon addition of both 1.6 mmol/l CoCl2 and 0.18 g/l bovine serum albumin to the assay solution at pH 5.6, the carboxypeptidase activity in human urine can be evaluated. Using our method, the daily excretion of carboxypeptidase in both healthy subjects and patients with renal diseases has been examined. The level is increased in patients with nephrotic glomerulonephritis (24.9 +/- 14.9 (SD) U/day; n = 8) and patients with nephritic glomerulonephritis (11.4 +/- 7.55 (SD) U/day; n = 14) as compared to that in healthy subjects (5.10 +/- 1.88 (SD) U/day; n = 18). However, it is decreased in patients with chronic renal failure (2.70 +/- 2.13 (SD) U/day; n = 12).

Cadmium↗

PubMed1983

Carboxypeptidase activity in the insulin secretory granule.

Carboxypeptidase activity was studied in subcellular fractions from a transplantable rat insulinoma and found to be localised principally in the insulin secretory granule. The activity, which was specific for peptide substrates with C-terminal basic amino acids, appeared to be a single enzyme with Mr 54000. This enzyme differed with respect to size and pH optimum from other basic amino acid-specific carboxypeptidases, such as carboxypeptidases B and N, and may be a secretory granule-specific enzyme involved in propolypeptide processing.

Adenoma, Islet Cell↗

PubMed1987

Hormone (pheromone) processing enzymes in yeast. The carboxy-terminal processing enzyme of the mating pheromone alpha-factor, carboxypeptidase ysc alpha, is absent in alpha-factor maturation-defective kex1 mutant cells.

Carboxy-terminal processing of the mating pheromone alpha-factor of the yeast Saccharomyces cerevisiae has been assumed to be due to the action of carboxypeptidase ysc alpha [(1985) EMBO J. 4, 173-177]. Here it is shown that a mutant (kex1) defective in alpha-factor maturation is defective in carboxypeptidase ysc alpha activity, indicating that the enzyme is indeed the processing catalyst. It is proposed that carboxypeptidase ysc alpha is the product of the KEX1 gene.

Carboxypeptidases↗

PubMed1990

Crystallization of serine carboxypeptidases.

Crystallization of three different serine carboxypeptidases has been achieved by the method of hanging-drop vapor diffusion. Serine carboxypeptidases II from wheat bran and malted barley crystallize isomorphously from polyethylene glycol solutions at room temperature (pH 4 to 7) in space group P4(1)2(1)2 or enantiomorph with cell dimensions of a = b = 98.2 A and c = 209.5 A. The crystals diffract to about 2.3 A resolution using rotating-anode X-ray generators. Assuming a dimer of Mr 120,000 in the asymmetric unit, Vm = 2.1 A3/dalton. These crystals appear suitable for structural studies. A genetically engineered serine carboxypeptidase from yeast, which lacks three of four glycosylation sites present in the wild-type, has also been crystallized by vapor diffusion against methylpentanediol at 4 degrees C, pH 6.4 to 8.0.

Carboxypeptidases↗

PubMed1983

Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase I.

We have studied the two vacuolar enzymes carboxypeptidase Y and aminopeptidase I from Saccharomyces cerevisiae with respect to biosynthesis, maturation and transfer from their site of synthesis into the organelle. The levels of translatable mRNA for these two proteins increase more than 10-fold at the end of the exponential growth period on glucose as carbon source and decrease again in the stationary phase. Two precursors of carboxypeptidase Y have been identified by in vivo pulse-labelling with [35S]methionine. These differ in their amount of carbohydrate as shown by inhibition of N-linked glycosylation with tunicamycin. The first is a protein with an apparent molecular weight of 67 kDa, which can be converted into the mature 60-kDa protein via an intermediate of 69 kDa. In the pep4-3 mutant, which is disturbed in the maturation of several vacuolar enzymes (Hemmings, B.A., Zubenko, G.S., Hasilik, A. and Jones, E.W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 435-439), the 69-kDa precursor accumulates in the vacuole. This suggests that the final proteolytic cleavage of carboxypeptidase Y can occur in the vacuole.

Aminopeptidases↗

PubMed1984

Carboxypeptidase-like activity during development and aging of Nippostrongylus brasiliensis.

An acid carboxypeptidase whose activity, based on several biochemical parameters, was determined to be similar to mammalian lysosomal carboxypeptidase was maximal at pH 5.0 during the early parasitic stages of development of the nematode Nippostrongylus brasiliensis. No such enzyme activity could be detected in the early free-living larval stages of this nematode. Carboxypeptidase activity declined as the age of the parasitic adult increased. Timing of maximum activity corresponds to the shift from free-living to parasitic stages of the life cycle.

Aging↗

PubMed1993

MK-801 and memantine protect cultured neurons from glutamate toxicity induced by glutamate carboxypeptidase-mediated cleavage of methotrexate.

Cleavage of methotrexate into glutamate and diaminomethylpteroate by intrathecal glutamate carboxypeptidase is a new approach to the treatment of acute methotrexate neurotoxicity. The simulation of glutamate carboxypeptidase rescue from high-dose methotrexate in neuron astrocyte cocultures of rat cerebellum or cerebral cortex resulted in a selective, concentration-dependent neurotoxicity. The neurotoxicity was caused by the enzymatic release of glutamate from methotrexate at lower concentrations of methotrexate, and by both glutamate and diaminomethylpteroate at concentrations of methotrexate exceeding 200 microM. The good neuroprotection afforded by MK-801 and memantine suggested that glutamate toxicity was mediated by N-methyl-D-aspartate receptors. Methotrexate alone was not toxic to astrocytes, neurons, or the neurite networking. [3H]thymidine and [3H]deoxyuridine incorporation studies showed that astrocyte proliferation in the presence of methotrexate was maintained by the reutilization of pyrimidine bases for DNA synthesis. N-methyl-D-asparate receptor antagonists should be coadministered in future experimental and clinical trials examining intrathecal glutamate carboxypeptidase rescue of methotrexate toxicity.

Animals↗

PubMed2004

Stereoselective inhibition of glutamate carboxypeptidase by organophosphorus derivatives of glutamic acid.

A series of alkyl and aryl phosphonyl, thiophosphonyl, and dithiophosphonyl derivatives of (S)- and (R)-glutamic acid were prepared and examined for inhibitory potency against glutamate carboxypeptidase (carboxypeptidase G). The acquisition of the phosphonamidodithioic acids and the individual phosphonamidothioic acid diastereomers was achieved through a common phosphonamidothiolate precursor, which also allowed for the chromatographic resolution of the chiral phosphorus center of the phosphonamidothioic acids. The most potent inhibitor of the series was the n-butylphosphonamidate derivative of the natural isomer of glutamic acid. Although each diastereomeric pair of three phosphonamidothionates exhibited stereoselective inhibition consistent with the configuration of the chiral phosphorus center, this effect was generally not remarkable. More important, was the effect of carbon stereochemistry upon glutamate carboxypeptidase inhibition as exemplified by a limited series of enantiomeric pairs of phosphonamidate and phosphonamidodithionate derivatives of glutamic acid. The phosphonamidate analogs derived from the unnatural stereoisomer of glutamic acid were devoid of inhibitory potency in contrast to their enantiomers. Surprisingly, the phosphonamidodithionates derived from the unnatural stereoisomer of glutamic acid demonstrated greater inhibitory potency than their naturally-derived antipodes.

Carboxypeptidases↗

PubMed2005

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori.

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.

Amino Acid Sequence↗

PubMed2005

Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae).

A full-length cDNA encoding a serine carboxypeptidase (designated SmSCP-1) was recovered from an ongoing salivary gland EST project of the wheat midge. The deduced 461-amino acid sequence had a putative signal sequence at the amino terminus, indicating it was a secreted protein. The protein shared homology with serine carboxypeptidases from other insects, mammals, plants, and yeasts. SmSCP-1 mRNA was expressed in all stages of development and detected in salivary gland and fat body tissues but not in midgut tissue. Expression analysis and quantitative real-time PCR assays in male and female wheat midges and the fat body tissue of adult midges revealed that SmSCP-1 was up-regulated nearly four-fold in the female midges compared to males and nearly two-fold in female fat body compared to male fat body. The wheat midge serine carboxypeptidase (SmSCP-1) most likely has a dual function. As a secreted digestive enzyme, it could play a role in mobilizing host-plant seed reserves for feeding larvae and as expressed in fat body could function as an exopeptidase in degradation of vitellogenin and/or in post-translational processing of other enzymes.

Amino Acid Sequence↗

PubMed2006

Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.

Amino Acid Motifs↗

PubMed2004

Carboxypeptidase N: a pleiotropic regulator of inflammation.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM (CK-MM). By removing only one amino acid, CPN has the ability to change peptide activity and receptor binding. CPN is a member of a larger family of carboxypeptidases, many of which also cleave arginine and lysine. Because of the highly conserved active sites and the possible redundant functions of carboxypeptidases, it has been difficult to elucidate the role of CPN in disease processes. The future use of gene ablation technology may be the most appropriate way to understand the function of CPN in vivo.

Amino Acid Sequence↗

PubMed2004

Serine carboxypeptidase-like acyltransferases.

In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of alpha/beta hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.

Acyltransferases↗

PubMed2003

Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi.

Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity. The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa. The enzyme has an optimum acidic pH (4.5 with furyl acryloyl-Phe-Phe as substrate), is highly specific for hydrophobic C-terminal amino acid residues, and is strongly inhibited by 3,4-dichloroisocoumarin (IC(50) value 0.3 microM). The enzyme is encoded by a number of genes arrayed in head-to-tail tandems; one of these genes has been cloned and sequenced. Sequence comparisons indicate that the enzyme belongs to the C group of serine carboxypeptidases, within the S10 serine peptidase family, and shows the higher similarity to plant and yeast enzymes. The residues involved in catalysis and most of those involved in substrate binding are conserved in the T. cruzi enzyme as well as 8 out of 10 Cys residues known to be involved in disulfide bridges in the yeast enzyme. This is the first report of an S10 family enzyme in trypanosomatids. The presence of serine carboxypeptidases is not restricted to T. cruzi, being possibly a general character of trypanosomatids.

Amino Acid Sequence↗

PubMed1977

[Characterization and separation of exocellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase and LD-carboxypeptidase from Bacillus sphaericus 9602].

Two exocellular enzymes have been characterized in the culture media of sporulating Bacillus sphaericus 9602 : a gamma-D-glutamyl-(L) meso-diaminopimelate endopeptidase and a L-lysyl-D-alanine carboxypeptidase. These two enzymes and the corresponding membrane-bound peptidases found in Bacillus sphaericus and Bacillus subtilis strains have similar activities. Their separation is described. Both enzymes were precipitated between 25 and 65 per cent (NH4)2SO4 saturation and a first chromatography was carried out on a column of DEAE-cellulose. The separation was performed by chromatography on hydroxyapatite, each enzyme was finally filtered through Ultrogel AcA 34. After separation, the endopeptidase activity and the carboxypeptidase activity increased respectively 93 and 11 fold. Both enzymes have a molecular weight near 200 000. By gel electrophoresis at pH 8.5, they were shown to have different mobilities : the carboxypeptidase is more anionic than the endopeptidase.

Alanine↗

PubMed1997

High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity.

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Animals↗

PubMed2002

Structural characterization of the human carboxypeptidase D gene and its promoter.

Human carboxypeptidase D (CPD) is a 180-kDa type I membrane protein with three tandem active site domains. CPD is a B-type (or kininase I-type) carboxypeptidase that cleaves C-terminal basic residues from proteins and peptides, such as Arg9 from bradykinin. The human carboxypeptidase D (CPD) gene was found to encompass approximately 88.3 kb of genomic sequence, containing 21 exons ranging in size from 65 to 1813 bp, and 21 introns ranging in size from 112 bp to 35.6 kb. Although CPD and CPM belong to the same metallocarboxypeptidase subfamily, their intron/exon structures differ significantly. Multiple transcription start sites were found in the CPD gene within a GC-rich sequence lacking the typical TATA box, but containing three GC boxes. Luciferase reporter assays with various size constructs containing the promoter region upstream of the start sites showed that it was active in three different cell lines, especially in the human hepatoma cell line HepG2 and the human monocytic cell line THP-1, which have high constitutive expression of CPD.

5' Flanking Region↗