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PubMed1983

Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida.

The gene coding for carboxypeptidase G2 was cloned from Pseudomonas sp. strain RS-16 into Escherichia coli W5445 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322. The plasmid isolated, pNM1, was restriction mapped, and the position of the gene on the 5.8-megadalton insert was pinpointed by subcloning. The expression of carboxypeptidase in E. coli was 100-fold lower than in the Pseudomonas sp. strain. When the cloned gene was subcloned into the Pseudomonas vector pKT230 and introduced into Pseudomonas putida 2440, a 30-fold increase in expression over that obtained in E. coli was observed. High expression (up to 5% soluble protein) was obtained in E. coli by subcloning a 3.1-megadalton Bg/II fragment into the BamHI site of pAT153. The increased expression was orientation dependent and is presumed to be due to transcriptional readthrough from the Tc promoter of the vector. Production of carboxypeptidase was shown to be induced (two-fold) by the presence of folic acid, and the mature protein was shown to be located in the periplasmic space of E. coli.

Carboxypeptidases↗

PubMed1989

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.

Base Sequence↗

PubMed1975

Carboxypeptidase B-like activity and C3 in cystic fibrosis.

Assays of carboxypeptidase B-like activity and C3 in serum from patients with cystic fibrosis and appropriate control subjects failed to demonstrate a deficiency of carboxypeptidase B-like activity or a consistent increase in C3, as was suggested by Conover and associates. Differences between men and women with cystic fibrosis were apparent, in that women with cystic fibrosis had higher concentrations of both serum carboxypeptidase B-like activity and C3 than either men with cystic fibrosis or control subjects.

Carboxypeptidases↗

PubMed2005

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1alpha in the circulation.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is an essential regulator of hematopoiesis, lymphocyte homing, pre-B-cell growth, and angiogenesis. As SDF-1alpha is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1alpha, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1alpha, leading to significant reduction in biologic activity, have not been identified. Using a new biochemical assay for measuring the carboxy-terminal cleavage activity, we purified from serum and plasma a peptidase that specifically removes the carboxy-terminal lysine from SDF-1alpha and identified it as carboxypeptidase N (CPN, also known as kininase I, arginine carboxypeptidase, and anaphylotoxin inactivator). We demonstrate that SDF-1alpha in serum and plasma lacks the carboxy terminal lysine, and depletion of CPN from serum and plasma significantly reduces the SDF-1alpha carboxypeptidase activity. Purified CPN effectively and specifically removes the carboxy-terminal lysine from SDF-1alpha and significantly reduces the chemokine's biologic activity as a pre-B-cell growth factor and chemoattractant. Thus, in addition to its role as a regulator of the biologic activity of kinins and anaphylatoxins, CPN is an important regulator of the biologic activity of SDF-1alpha by reducing the chemokine-specific activity.

Animals↗

PubMed1993

Generation of 1-37 and 1-38 forms of adrenocorticotropin by mono- and dipeptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles.

ACTH is a 39-amino acid peptide synthesized in the pituitary as part of the precursor molecule, POMC. Analysis of bovine anterior pituitary homogenates and secretory vesicles revealed that in addition to ACTH-(1-39), ACTH-(1-37) and ACTH-(1-38) were also present in the lobe, indicating that carboxyl-terminal processing of ACTH-(1-39) occurred in vivo. Mono- and dipe;tidyl carboxypeptidase activities that cleaved ACTH-(1-39) were detected in bovine intermediate and anterior pituitary secretory vesicle membranes and characterized. The dipeptidyl carboxypeptidase activity liberated ACTH-(1-37) and the dipeptide, Glu-Phe, and the monocarboxypeptidase activity generated, to a smaller extent, ACTH-(1-38) and phenylalanine from ACTH-(1-39). Kinetic studies indicated that the formation of ACTH-(1-37) occurred within minutes, whereas the formation of ACTH-(1-38) occurred within hours. Both enzymatic activities had a pH optimum of 5.5 and a Km of 14-18 microM for ACTH-(1-39), and were inhibited by serine and some thiol, but not metallo- or aspartic protease inhibitors. These unique serine carboxypeptidase(s) may function as a converting enzyme in vivo.

Adrenocorticotropic Hormone↗

PubMed2002

Down-regulation of aortic carboxypeptidase-like protein during the early phase of 3T3-L1 adipogenesis.

Aortic carboxypeptidase-like protein (ACLP) is a 175-kDa protein that is expressed in vascular smooth muscle cells and contains a signal peptide sequence, a lysine- and proline-rich repeating motif, a discoidin-like domain with 35% identity to discoidin I, and a carboxypeptidase-like domain that is 39% identical with carboxypeptidase E. It is secreted into the extracellular matrix and may play a role in abdominal wall development and dermal wound healing. ACLP is also expressed in adipose tissue, but at lower levels. In this study we demonstrate that ACLP protein and mRNA are severely down-regulated in the early phase of 3T3-L1 preadipocyte differentiation induced by insulin, dexamethasone, and isobutylmethylxanthine. Neither dexamethasone, isobutylmethylxanthine, nor insulin treatment alone reduced the level of ACLP protein, suggesting that ACLP down-regulation is a differentiation-associated event. ACLP down-regulation coincided with the onset of the postconfluent mitotic clonal expansion phase of adipogenesis. In contrast, subconfluent 3T3-L1 cell proliferation did not alter ACLP expression, suggesting a specific linkage between ACLP and differentiation-induced clonal expansion. Stable overexpression of ACLP had no effect on preadipocyte differentiation assessed by triacylglycerol accumulation and peroxisome proliferator-activated receptor-gamma levels. The role of ACLP and its marked reduction during adipogenesis merit further study.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Purification and characterization of serine carboxypeptidases from Absidia zychae.

Among 29 strains of zygomycetes screened for serine carboxypeptidases, Absidia zychae NRIC 1199 showed the highest enzyme production. Two serine carboxypeptidases, CPZ-1 and CPZ-2, were purified to a homogeneous state from an extract of koji culture of A. zychae NRIC 1199. Purified CPZ-1 and CPZ-2 showed similar properties except the isoelectric point (pI); The pI of CPZ-1 and CPZ-2 were 4.50 and 4.65, respectively. The molecular weights of the CPZ-1 and CPZ-2 were 48,000 by SDS-PAGE and gel filtration. Among the proteinase inhibitors tested, phenylmethylsulfonyl fluoride and monoiodoacetic acid strongly inhibited the enzyme activity. The optimum pHs of CPZ-1 and CPZ-2 were 4.2 towards Z-Glu-Tyr. It is shown that the substrate specificities of CPZ-1 and CPZ-2 were dependent on the presence of bulky amino acid residues in the penultimate position (P1) for the small Z-peptides. However, in spite of the presence of Gly, Asp, Arg, or Pro in the P1 position, oligopeptides were hydrolyzed rapidly. CPZ-1 and CPZ-2 had not only carboxypeptidase but also carboxyamidase and amidase activities, and acted preferentially as a carboxyamidase for C-terminal amidated peptides. The hydrophobicity of P2 and P3 positions and the bulkiness of P1 and P'1 positions of the substrate may be important for carboxyamidase and amidase activities.

Amino Acid Sequence↗

PubMed2003

Synthesis of hydroxymethylglutathione from glutathione and L-serine catalyzed by carboxypeptidase Y.

Hydroxymethylglutathione (gamma-L-glutamyl-L-cysteinyl-L-serine; hmGSH) occurs in many species belonging to the family Gramineae, but the biosynthetic pathway for hmGSH has not been identified. We found that carboxypeptidase Y (CPY), but not carboxypeptidase A, catalyzed hmGSH synthesis from glutathione and L-serine in vitro at acidic pH. CPY also catalyzed methylglutathione synthesis from glutathione and L-alanine. These findings suggested that a carboxypeptidase-like enzyme may be involved in hmGSH synthesis in vivo.

Alanine↗

PubMed1979

Studies of the specific role of the subunits of choriogonadotropin for biological, immunological and physical properties of the hormone. Digestion of choriogonadotropin and its isolated subunits with serine carboxypeptidase.

The residues 90-92 can be split off from the C-terminal region of the isolated alpha-subunit of choriogonadotropin (residues 88--92: -Tyr-Tyr-His-Lys-Ser-OH) by means of serine carboxypeptidase (des-Lys91,Ser92-alpha-subunit; des-(90-92)-alpha-subunit). However, when choriogonadotropin is digested by serine carboxypeptidase, only the residues 143-145 (-Leu-Pro-Gln-OH) form the C-terminus of the beta-subunit are released (des-(143-145)-choriogonadotropin). Depending on the pH conditions, glutamine 145 and the residues 143-145, respectively, are liberated by digestion of the isolated beta-subunit (des-Gln145-beta-subunit and des-(143-145)-beta-subunit, respectively). The present study provides evidence that the C-termini of both the isolated subunits and those in choriogonadotropin are probably arranged on the surface of the molecules. The biological activity of des-(143-145)-choriogonadotropin is not significantly decreased. The immunological activity, however, is reduced when measured by complement fixation. In comparison to the native hormone, a four-fold amount of des-(143-145)-choriogonadotropin has to be applied to obtain highest complement fixation. The conformation of des-(143-145)-choriogonadotropin does not seem to differ from that of the native hormone, when estimated both by CD measurements and by Ans-choriogonadotropin fluorescence. The respective determinant therefore seems to depend, at least to some extent, on the sequence of the C-terminal region of the beta-subunit of the hormone; complement fixation, however, does not seem to be affected significantly, when the des-(143-145)-beta-subunit is compared with the native beta-subunit using an antiserum against the native beta-subunit. This provides evidence that this C-terminal determinant is possibly more immunogenic at the hormone than at the isolated beta-subunit. The biological activity of recombined choriogonadotropin in vivo as well as in vitro is markedly reduced when serine 92 is removed from the C-terminus of the alpha-subunit (des-Ser92,Lys91-alpha-native beta-subunit: 36% residual activity in vivo). Biological activity is lost when the residues 88-90 are removed by digestion of the des-Ser92,Lys91-alpha-subunit with carboxypeptidase A. Recombination products between a modified alpha-and the native beta-subunit show a reduced Anschoriogonadotropin fluorescence (des-Lys91,-Ser92-alpha + native beta-subunit: 52%; des-(88-92)-alpha- + native beta-subunit: 23%). The Ans-induced aggregation of choriogonadotropin, however, also takes place in those recombination products which display a low Ans-choriogonadotropin fluorescence, indicating that the reduction is probably not caused by a portion of the molecules losing their binding sites for Ans. Therefore the diminished Ans-choriogonadotropin fluorescence seems to signal small conformational changes. The CD spectra of the native and the des-(90-92)-alpha-subunit, however, seem not to differ significantly. It is shown that the release of amino acids from the C-terminus of the alpha-subunit causes a disturbance of the interaction between the subunits. This seems to prevent an effective conformational change of the beta-subunit which probably is a prerequisite for the binding of the hormone to the receptors of Leydig cells.

Amino Acid Sequence↗

PubMed1979

Characterization of an intracellular inhibitor of the carboxypeptidase R from Rhodotorula glutinis.

A peptidic inhibitor of the carboxypeptidase R from Rhodotorula glutinis has been identified and partially purified. A molecular weight of 31 000 was found by gel filtration. The inhibitor is reversibly separated from the carboxypeptidase by chaotropic agents. Removal of the inhibitor by an acid protease explains the osberved activation of the carboxypeptidase by incubation at acidic pH values.

Carboxypeptidases↗

PubMed2003

Expression and distribution of carboxypeptidase B in the hippocampal subregions of normal and Alzheimer's disease brain.

Earlier neurochemical studies suggested that human brain carboxypeptidase B may play a significant role in the degradation of amyloid-beta1-42 in the brain. Using an immimohistochemical technique we report here on the neuronal expression and distribution of this enzyme in the segments (CA1a, CA1b and CA1c) of the CA1 subfield and in area CA4 of the hippocampus in normal and Alzheimer's disease brain samples. Its distribution was compared with the appearance of neurofibrillary tangles in the same brain sample. For immunohistochemical localization of carboxypeptidase B, a specific C14-module antibody was applied, together with the Gallyas silver impregnation technique for the demonstration of neurofibrillary tangles. The results revealed that, in the control samples, most of the immunoreactivity appeared in segment CA1a in the pyramidal cells, less in segment CA1b and least in segment CA1c. In the Alzheimer's disease samples, there was no particular immunostaining in the neurons, but, a large number of silver-impregnated degenerated neurons appeared. The results support the suggestion that carboxypeptidase B may play a significant role in elimination of the intracellular accumulation and toxicity of amyloid-beta in the human brain and thereby protect the neurons from degeneration.

Aged↗

PubMed1996

Piperastatin A, a new selective serine carboxypeptidase inhibitor produced by actinomycete. I. Taxonomy, production, isolation and biological activities.

Piperastatin A (structure, N-formyl-allo Ile-Thr-Leu-Val-Pip-Leu-Pip, Pip = hexahydropyridadine-3-carboxylic acid; molecular weight, 809), a new inhibitor of serine carboxypeptidase was discovered in the fermentation broth of Streptomyces lavendofoliae MJ908-WF13. It was purified by activated charcoal chromatography, YMC gel ODS-A chromatography and centrifugal partition chromatography (CPC) by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y), and finally obtained as colourless needles. Piperastatin A is a competitive inhibitor of the enzyme with Ki = 52 +/- 6.2 nM. Piperastatin A is a highly specific inhibitor of the serine carboxypeptidases, CP-Y and platelet deamidase with little effect on related enzymes, has no antimicrobial activity and has low toxicity.

Carboxypeptidases↗

PubMed2004

Microbial serine carboxypeptidase inhibitors--comparative analysis of actions on homologous enzymes derived from man, yeast and wheat.

The actions of peptidase inhibitors derived from Streptomycete on human cathepsin A (hCath A), yeast carboxypeptidase Y (CPY), and wheat carboxypeptidase II (CPW) were analyzed comparatively. Lactacystin and omuralide (clasto-lactacystin beta-lactone), well-known cytoplasmic proteasome inhibitors, both had a potent and non-competitive inhibitory effect on these homologous serine carboxypeptidases, although they inhibited CPW and hCath A more effectively than CPY in vitro. Ebelactone B exhibited a mixed non-competitive inhibitory effect and selectivity for CPY. Piperastatin A showed competitive inhibition of CPY and hCath A but had little effect on CPW. In contrast, chymostatin inhibited CPW efficiently, while it had less effect on hCath A and CPY. In cell culture system, lactacystin was the most potent as to inactivation of the intralysosomal recombinant hCath A activity expressed in a genetically engineered fibroblastic cell line with galactosialidosis (hCath A deficiency). These results suggest that the specific inhibitory effects of lactacystin and its derivatives on hCath A might be applicable to elucidate the pathophysiological roles in the human deficinecy.

Carboxypeptidases↗

PubMed2001

An immunocytochemical approach to the demonstration of intracellular processing of mast cell carboxypeptidase.

Newly synthesized secretory proteins are transported from the rough endoplasmic reticulum to the Golgi complex where they can undergo posttranslational modification and are then packaged for secretion by concentration within membrane-bound very small progranules that fuse to form large immature granules. The contents of these vesicles are thought to be then processed, forming mature secretory granules. After acquiring their mature appearance, the secretory granules reside in the cytoplasm until they are secreted. In this study, we raised antibodies against the first 15 N-terminal amino acids of mast cell pro-carboxypeptidase and the last 14 C-terminal amino acids of mast cell carboxypeptidase. Immunohistochemical localization of the two peptides was carried out in human breast tissue and rat tissue (ear, skin, peritoneum, and tongue). In all cases, both epitopes were demonstrated only in mast cell secretory granules. However, mast cells from 3-week-old rats were more positive for the pro-enzyme compared to 3-month-old rats. Human mast cells in breast tissues were mostly negative for the pro-enzyme and positive for the carboxypeptidase. On the basis of these observations, it seems that posttransitional modification of the pro-enzyme to form the active enzyme occurs in the mast cell secretory granules.

Amino Acid Sequence↗

PubMed1976

Purification to homogeneity and properties of two D-alanine carboxypeptidases I From Escherichia coli.

Three homogeneous preparations of D-alanine carboxypeptidases I have been obtained from Escherichia coli strain H2143, termed enzymes IA, IB, and IC. Enzyme IA purified from the membrane after extraction with Triton X-100 appeared on sodium dodecyl sulfate gel electrophoresis to be a polypeptide doublet whose monomer molecular weights were about 32,000 and 34,000. In addition to D-alanine carboxypeptidase activity, it catalyzed a transpeptidase reaction with several substrates, bound [14C]penicillin G, had a weak penicillinase activity, but was devoid of endopeptidase activity. Enzyme IB obtained from the membrane after LiCl extraction and enzyme IC obtained from the supernatant solution were either identical or extremely similar. They were composed of a single polypeptide whose monomer molecular weight was about 41,000. In addition to carboxypeptidase activity, they catalyzed an endopeptidase reaction, had weak penicillinase activity, and had very poor transpeptidase activity, but did not bind [14C]penicillin G. Some data relating to the mechanism of catalysis by these enzymes are described. Their possible physiological role is discussed.

Binding Sites↗

PubMed1992

[Purification and physico-chemical properties of soluble carboxypeptidase N from cat brain gray matter].

Using affinity chromatography on diasorb-L-arginine and polyacrylamide gel electrophoresis, soluble carboxypeptidase H (E. C. 3.4.17.10) has been isolated from cat brain cortex and purified 598-fold with a 16% yield. The enzyme has a molecular mass of 50 kDa, consists of one polypeptide chain, and displays the maximum activity at pH 5.6. Carboxypeptidase H is a thiol-dependent metalloenzyme and contains a Zn2+ ion in its active center. The Km and V values for dansyl-Phe-Leu-Arg are 100 +/- 5 microM and 12.5 +/- 1.4 microM/min/mg of protein, respectively. The existence of two forms of soluble carboxypeptidase differing in isoelectric points and pH optima has been demonstrated. The enzyme with a pI of 4.8 has a pH optimum at 5.5-5.6, while that with a pI of 5.25-at 6.0.

Animals↗

PubMed2005

[Lysosomal carboxypeptidase A].

Lysosomal carboxypeptidase A (cathepsin A) is synthetized in the form of preproenzyme, which undergoes to active enzyme as a result of post-translational modification. It splits off C-terminal amino acid residues from peptides and proteins and synergizes with other proteases in degradation of cellular proteins in lysosomes. Lysosomal carboxypeptidase A has an effect on peptide hormones and peptides of biological activity of tissues and body fluids as well. It forms complexes with some glycosidases that protects them against proteolytic degradation. Deficiency of this enzyme induces storage diseases. Lysosomal carboxypeptidase A as multifunctional enzyme plays an important regulatory role in organismal metabolism.

Amino Acid Sequence↗

PubMed2006

[The effect of prenatal stress on the carboxypeptidase H activity in the hypothalamo-pituitary-adrenal-gonadal system of the rat].

Effects of dams stress on the carboxypeptidase H: the neuropeptide exchange enzyme in the hypothalamo-pituitary-adrenall-gonadal system, was studied in the litter of different age (0, 14, 28, 45, and 120 day after birth) was studied. The sex differences in dynamic of enzyme activity in intact animals are substantiated. The effect of prenatal stress on carboxypeptidase H activity dependes on age and sex of animals. Prenatal stress is altering during the age dynamics of enzyme activity. This in the pituitary gland and hypothalamus of prenatal stressed female rats was a control for the male rats. In the adrenal and gonadal gland of prenatal stressed males, this was a control for the female rats female. The role of carboxypeptidase H in pubescence and mechanisms of effect of prenatal stress on sex system functional are discussed.

Animals↗