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At least 667 records · Page 37Linked to original sources
PubMed1981

Enzymatic properties of an acid carboxypeptidase from Aspergillus niger var. macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) isolated from Aspergillus niger var. macrosporus was investigated in regard to its kinetic parameters for two synthetic substrates. The optimum pH of peptidase activity toward Z-Glu-Tyr-OH was pH 3.0 Km and kappa cat values were 4.0 . 10(-3) M and 270 s-1 at pH 3.0 and 30 degrees C. The optimal pH of esterase activity toward Bz-Arg-OEt was pH 5.2 Km and kappa cat for esterolytic activity were 6.1 . 10(-4) M and 1500 s-1 at pH 5.0 and 30 degrees C. The enzyme released expected amino acids sequentially from the carboxyl ends of S-beta-aminoethylated ribonuclease A and the B-chain of oxidized insulin, demonstrating carboxypeptidase activity of the enzyme. The enzyme was inactivated by diisopropylphosphorofluoridate and phenyl-methanesulfonyl fluoride. In the reaction with [14C]diisopropylphosphorofluoridate, the amount of [14C]diisopropylphosphoryl group incorporated into the enzyme in complete inactivation was estimated as 2 mol/mol enzyme.

Aspergillus niger↗
PubMed1990

Deficient lysosomal carboxypeptidase activity in galactosialidosis.

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).

Carbohydrate Metabolism, Inborn Errors↗
PubMed1992

Cleavage of farnesylated COOH-terminal heptapeptide of mouse N-ras by brain microsomal membranes: evidence for a carboxypeptidase which specifically removes the COOH-terminal methionine.

Brain microsomal membranes are capable of sequentially removing Met, Leu and Val from a chemically synthesized COOH-terminal heptapeptide (propionyl-Gly-Ser-Pro-(farnesyl-Cys)-Val-Leu-Met) of mouse N-ras protein. The carboxypeptidase generating Met displays maximum activity at neutral pH and shows high affinity for the farnesylated substrate (Km = 73 microM) as compared to its non farnesylated precursor (Km = 600 microM). The results of inhibitor action suggest that the membrane carboxypeptidase is a novel, probably thiol-dependent, serine type peptidase.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS.

A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp13 library by complementation of the previously characterized mutation cps1-1 [(1981) J. Bacteriol. 147, 418-426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N-terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxy-peptidase yscS as sole nitrogen source.

Amino Acid Sequence↗
PubMed1994

Substrate specificity of Escherichia coli LD-carboxypeptidase on biosynthetically modified muropeptides.

Escherichia coli murein can be biosynthetically modified. Amino acids at positions 3 and 4 (m-diaminopimelic acid and D-alanine, respectively) on the peptide moieties can be changed under appropriate growth conditions. The activity of E. coli LD-carboxypeptidase on biosynthetically modified substrates has been studied in vitro. The enzyme hydrolysed all tested disaccharide-tetrapeptide monomeric muropeptides modified at position 4. Monomers with m-lanthionine, but not with L-ornithine, instead of m-diaminopimelic acid at position 3 were accepted. However, neither cross-linked muropeptides nor macromolecular murein were substrates for the reaction. Our observations argue against a direct effect of LD-carboxypeptidase on macromolecular murein metabolism.

Alanine↗
PubMed1992

Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.

Animals↗
PubMed1976

Subcellular localization of gamma-glutamyl carboxypeptidase and of folates.

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [3H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions. The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H] folic acid was initially concentrated in the microsomal fraction, as measured by 3h cpm per ng folate. Initially, injected [3H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total 3H cpm determined to be [3H] folate penta- and hexaglutamates. At 24 h, the conversion of [3H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H] folic acid after 2h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.

Acid Phosphatase↗
PubMed1992

Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147.

Cloning and nucleotide sequencing identified the vanY gene as a member of the vancomycin-resistance van gene cluster of enterococcal plasmid, pIP816. The vanY gene was necessary for synthesis of the vancomycin-inducible D,D-carboxypeptidase activity previously proposed to be responsible for glycopeptide resistance. However, this activity was not required for peptidoglycan synthesis in the presence of glycopeptides. The deduced product of vanY did not display significant similarity with other D,D-carboxypeptidases.

Amino Acid Sequence↗
PubMed1992

Expression of a synthetic gene encoding potato carboxypeptidase inhibitor using a bacterial secretion vector.

A synthetic gene encoding the 39-amino-acid (aa) potato carboxypeptidase inhibitor IIa (PCI-IIa) has been constructed and expressed using the secretion vector, pIN-III-ompA-3, fused in frame to the OmpA signal peptide-encoding sequence. Recombinant Escherichia coli secreted a PCI with 10 additional aa at the N terminus (rePCI + 10). These extra aa were removed by site-directed mutagenesis giving a PCI with no additional aa (rePCI), as shown by fast atom bombardment mass spectrometry (M(r) 4295). The two forms of rePCI were found almost exclusively in the culture medium, not in the periplasmic space, as would be expected from OmpA signal peptide fusions. Both rePCI + 10 and rePCI are biologically active and react strongly with serum raised against PCI from potato. A method for the purification of rePCI to homogeneity has been developed. The purified rePCI shows a Ki for carboxypeptidase A within the range of the natural PCI-IIa (1.5-2.7 nM). These results indicate that both rePCI + 10 and rePCI are properly folded and that their three disulfide bridges are correctly formed. Together with previous reports, our results show that fusion to a secretion signal peptide is an effective way of producing small proteins containing disulfide bridges in a biologically active form.

Amino Acid Sequence↗
PubMed2006

A rapid and sensitive assay for the quantitation of carboxypeptidase N, an important regulator of inflammation.

BACKGROUND: Carboxypeptidase N is a plasma zinc metallocarboxypeptidase which is constitutively expressed in the liver and was identified as the enzyme responsible for inactivating bradykinin and kallidin by removing the C-terminal arginine. Because CPN can cleave the C-terminal arginine of C3a, C4a and C5a it is often referred to as anaphylatoxin inactivator. Markedly reduced levels of circulating CPN are associated with recurrent angioedema and abnormal cutaneous polymorphonuclear cell infiltration. METHODS: In this paper we describe a fast kinetic coupled enzymatic assay for the sensitive measurement of carboxypeptidase N activities in serum samples. The assay makes use of the excellent CPN substrate Benzoyl-L-Alanyl-L-Arginine. RESULTS: This novel assay is very fast, easy to perform and combines good reliability and reproducibility with excellent correlation with the HPLC-assisted assay (r=0.927; n=140). CONCLUSION: The presented assay can be used for high throughput screening of this important regulator of inflammation in clinical plasma or serum samples.

Adolescent↗
PubMed2005

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond.

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19-Cys43 disulfide bond that links the beta-sheet core with the alpha-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2A resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by approximately 1.5 kcal mol(-1) (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity.

Animals↗
PubMed2003

Localization of carboxypeptidase A-like enzyme in rat kidney.

In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 degrees C, pH 7.4, were Ang 1-9 and Ang II. Quinaprilat did not influence the formation of Ang 1-9, but it inhibited formation of Ang II. The formation of Ang 1-9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1-9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.

Angiotensin I↗
PubMed2005

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relationship to cystatins and the tumor suppressor TIG1.

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Amino Acid Motifs↗
PubMed1995

Human nasal mucosal carboxypeptidase: activity, location, and release.

BACKGROUND: Carboxypeptidases (CPs), such as carboxypeptidase N (CPN) (kininase I, E.C.3.4.17.3), may regulate peptide-mediated vasodilation and vascular permeability in respiratory mucosa by degrading proinflammatory peptides such as bradykinin, anaphylatoxins, and neuropeptides during allergic and nonallergic inflammation. The sources of CP activity in human nasal secretions were investigated. METHODS: Well-characterized human nasal provocation and secretion analysis methods were used. Potential sources of CPN in human nasal mucosa were identified by immunohistochemistry. CP activity was defined as DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid inhibitable Bz-Gly-Lys degradation. CP activity was measured in nasal mucosal homogenates and nasal lavage fluids induced by methacholine, histamine, and allergen nasal provocation. RESULTS: CPN-immunoreactive material was localized to the glycocalyx of the epithelium, some vessels, and gland ducts near the epithelial basement membrane but not to submucosal gland cells. CP activity in human nasal lavage fluid after saline nasal provocation was 0.10 +/- 0.04 U/L. Histamine provoked secretion of significantly more CP activity (3.84 +/- 0.99 U/L; p < 0.01 vs saline). Methacholine did not significantly increase secretion (0.54 +/- 0.22 U/L). After nasal allergen challenge, CP activity was at a maximum between 11 and 20 minutes, and CP activity correlated with IgG concentration (r = 0.91, p < 0.01), a marker for proteins of plasma origin, suggesting that CP activity originated in plasma. CONCLUSIONS: These data suggest that plasma is the predominant source of CP activity secreted from human nasal mucosa and that plasma extravasation and interstitial fluid exudation across the epithelium are the primary processes regulating its appearance in nasal secretions.

Adult↗
PubMed1997

Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidases in human adrenal tumors.

Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidase activities were measured in the supernatant (S2) and pellet (P2) fractions obtained by ultracentrifugation of human adrenal tumor preparations. Negligible enzyme activity was found in cortical tumor whereas highly significant activities were present in the P2 fractions of the two pheochromocytoma specimens. The hydrolysis rates, expressed in terms of the percent of added substrate were 58-66%/60 min for Hip-Phe-Arg, 55-58%/60 min for Hip-Arg-Phe and 19-30%/60 min for Hip-His-Leu. The angiotensin-converting enzyme inhibitor, captopril, differentially inhibited the enzyme splitting Hip-His-Leu versus the one cleaving Hip-Arg-Phe; Hip-Phe-Arg is probably the substrate of both. It is concluded that the Hip-Arg-Phe-cleaving enzyme in adrenomedullary tumor is probably identical to the purportedly novel dipeptidyl carboxypeptidase that we detected earlier in rabbit ear artery wall, which converts (Met5)-enkephalin-Arg6,Phe7 to (Met5)-enkephalin.

Adrenal Gland Neoplasms↗
PubMed1998

The amino acid sequences of carboxypeptidases I and II from Aspergillus niger and their stability in the presence of divalent cations.

The amino acid sequences of serine carboxypeptidase I (CPD-I) and II (CPD-II), respectively, from Aspergillus niger have been determined by conventional Edman degradation of the reduced and vinylpyridinated enzymes and peptides hereof generated by cleavage with cyanogen bromide, iodobenzoic acid, glutamic acid cleaving enzyme, AspN-endoproteinase and EndoLysC proteinase. CPD-I consists of a single peptide chain of 471 amino acid residues, three disulfide bridges and nine N-glycosylated asparaginyl residues, while CPD-II consists of a single peptide chain of 481 amino acid residues, has three disulfide bridges, one free cysteinyl residue and nine glycosylated asparaginyl residues. The enzymes are closely related to carboxypeptidase S3 from Penicillium janthinellum. Both Ca2+ and Mg2+ stabilize CPD-I as well as CPD-II, at basic pH values, Ca2+ being most effective, while the divalent ions have no effect on the activity of the two enzymes.

Amino Acid Sequence↗
PubMed1981

Conversion of Met-enkephalin-Arg6-Phe7 by a purified brain carboxypeptidase (cathepsin A).

A carboxypeptidase A-like enzyme known as cathepsin A was purified from rat brain by extraction with Triton X-100, followed by chromatography on DEAE-Sephadex A-50 and gel-filtration. Purified enzyme was devoid of contamination of tryptic-like enzymes, by dipeptidyl carboxypeptidase (angiotensin converting enzyme) and of enkephalinnases cleaving the Tyr-Gly and Gly-Phe bonds of Met-enkephalin. Incubation of purified enzyme with Met-enkephalin-Arg6-Phe7, a naturally occurring enkephalin surrogate, was accompanied by the release of three products as detected by reverse phase HPLC. Subsequent amino acid analysis identified these as Phe, Met-enkephalin-Arg6, and Met-enkephalin, indicating cleavage at the Arg6-Phe7 and Met5-Phe6 bonds. Breakdown followed a precursor-product-relationship with the hexapeptide appearing as an intermediate and the pentapeptide as the final product. The Km for cleavage of the Arg-Phe site was 0.09 mM. Rates of cleavage of hexa- and heptapeptide accord with those found for synthetic N-protected dipeptide substrates. Cathepsin A does not act as an enkephalinase in the accepted sense, since no breakdown of Met-enkephalin was observed.

Amino Acid Sequence↗