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PubMed1989

cDNA cloning and complete primary structure of the small, active subunit of human carboxypeptidase N (kininase 1).

The human plasma metallo-protease carboxypeptidase N of Mr 280,000 consists of two small, enzymatically active subunits of Mr 50,000 and two large subunits. Only the large subunits are glycosylated. They may have a function in stabilizing the complex in plasma. The N-terminal sequence of the small subunit was determined from the isolated protein and used to specify a unique 59-mer oligonucleotide probe. A cDNA clone of 1.7 kbp containing the entire coding sequence of the small subunit of carboxypeptidase N was isolated from a human-liver cDNA library. The cDNA clone encodes a signal sequence of 20 amino acids and the 438 amino acids of the mature subunit. There is a remarkable primary structure similarity of 49% to bovine carboxypeptidase E (enkephalin convertase). A more distant relationship to the bovine pancreatic, digestive carboxypeptidases A and B or even to the metallo-endopeptidases is based mainly on the occurrence of conserved, mechanistically important residues.

Amino Acid Sequence↗

PubMed1985

Differential distribution of carboxypeptidase-processing enzyme activity and immunoreactivity in membrane and soluble components of chromaffin granules.

A comparison of carboxypeptidase-processing enzyme activity and immunoreactivity showed that they were differentially distributed in soluble and membrane components of bovine adrenal medulla chromaffin granules. The majority of enzyme activity (80% of total activity in the granules) was present in the soluble fraction, but the number of enzyme molecules was distributed equally between the soluble and membrane fractions. When equivalent amounts of carboxypeptidase enzyme (ng of immunoreactivity) in each fraction were compared, the carboxypeptidase in the soluble component appeared to be five to six times more active than the membrane-bound form of the enzyme. The soluble and membrane components of the granules may represent populations of enzyme at different states of activation. This finding could have important implications for the regulation of the carboxypeptidase-processing enzyme.

Animals↗

PubMed1979

Effect of cephapirin on formation of D-alanine carboxypeptidase in growing Bacillus subtilis cells.

Cephapirin was utilized to examine the interaction of beta-lactam antibiotics with growing Bacillus subtilis cells and the biological effects simultaneously produced. Saturation binding and quantitative cell death were observed at the cephapirin concentration of 0.1 mug/ml. Cephapirin bound to all penicillin-binding proteins except the d-alanine carboxypeptidase. A specific [(14)C]benzylpenicillin-binding assay was developed for the d-alanine carboxypeptidase. At the lowest saturating concentration of antibiotic (0.1 mug/ml), cephapirin inhibited formation of the d-alanine carboxypeptidase. Upon incubation with cephapirin, 18% of the membranous d-alanine carboxypeptidase was released into the media. The data suggest that beta-lactam antibiotics may affect the formation of bacterial cytoplasmic membranes in addition to their effect on cell wall synthesis.

Bacillus subtilis↗

PubMed1998

Immunohistochemical localization of carboxypeptidases E and D in the human placenta and umbilical cord.

Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins.

Carboxypeptidase H↗

PubMed1991

Identification and cloning of a granule autoantigen (carboxypeptidase-H) associated with type I diabetes.

Using serum from a prediabetic patient as a probe, we screened 0.5 x 10(6) recombinants from a rat islet lambda gt11 expression library. One plaque-producing antigen reactive with this prediabetic serum was identified, subcloned, and sequenced. Analysis of the sequence reveals that the clone encodes a 136-amino acid fragment of carboxypeptidase-H (enkephalin convertase). Carboxypeptidase-H is a molecule expressed within islet secretory granules and neurendocrine cells. The patient whose antibodies recognize this recombinant molecule (termed DG-1) was negative for anti-DG-1 antibodies in 1984, developed the antibodies by 1986, and remained positive until the development of diabetes in 1988. To date, serum from 5 of 20 cytoplasmic islet cell antibody-positive relatives reacted with the expressed protein, while none of 14 control sera reacted. On Western blotting, the initial patient's serum used for the screening reacts with a 52-kDa antigen corresponding to the mol wt of the membrane form of carboxypeptidase-N. The current study has identified carboxypeptidase-H as an autoantigen recognized by serum of pretype I diabetes, and the methodology used should aid in identification of additional autoantigens associated with type I diabetes.

Antibodies↗

PubMed1983

A simple spectrophotometric assay of carboxypeptidase N (kininase I) in human serum.

Kininase I (carboxypeptidase N; EC 3.4.17.3) consists of carboxypeptidase N1 (CN1) and carboxypeptidase N2 (CN2); these two enzymes can be differentiated by their activities towards hippuryl-L-arginine and hippuryl-L-lysine, respectively. A spectrophotometric assay for both carboxypeptidases in human serum is described and the biochemical behaviour of these enzymes investigated. The pH optima are found to be 8.4 for CN1 and CN2. The Michaelis-Menten constants are: CN1 4.59 +/- 0.03 mmol/l; CN2 37.26 +/- 3.49 mmol/l. CN2 can be inhibited by EDTA (76%), dimercaprolum (97%) and phenanthroline (98%). Diisopropylfluorophosphate has no influence on both enzymes. Elevated haemoglobin only interferes with CN1 measurements, and high bilirubin concentrations slightly alter the activity of both enzymes. High CN1 activities were found in sera of patients with sarcoidosis, and elevated CN2 activities were found in lung cancer.

Arginine↗

PubMed1980

Familial carboxypeptidase N deficiency.

Carboxypeptidase N is a serum metalloenzyme that inactivates C3a, C4a, C5a, bradykinin, kalladin, and fibrinopeptides. Of 172 sera from patients with chronic urticaria or angioedema, one had a remarkably depressed carboxypeptidase N level (21% of normal). Of sera from 103 patients with other diseases, elevated levels were observed in cases of neoplasms, and one abnormally low value was detected in a patient with cirrhosis. The patient with a remarkably low carboxypeptidase N level was a 65-year-old man with an 11-year history of episodic angioedema occurring about 40 times per year. Inactivation of C3a and lysyl-bradykinin by his serum was markedly prolonged. Plasma histamine was elevated during attacks, but serotonin and kinin activity were not. The proband's sister had an equally depressed serum carboxypeptidase N level, and studies of other family members suggested an autosomal recessive inheritance of the enzyme deficiency.

Aged↗

PubMedDate not supplied

[Effect of chloroform and ether narcosis on the activity of basic carboxypeptidases in the hypothalamic-pituitary-adrenal axis in rats].

It is discovered that chloroform narcosis does not influence on carboxypeptidase H and phenylmethylsulfonyl fluoride-inhibited carboxypeptidase activity in the rats hypothalamic-pituitary-adrenal axis. Ether narcosis provokes an increase of PMSF-inhibited carboxypeptidase activity in the pituitary body approximately in 8 times and carboxypeptidase H activity in hypothalamus by 29 percents in comparison with the intact animals. It is supposed that at research neuropeptides and their metabolism enzymes and especially the answer to a stress chloroform narcosis would be the better anaesthesia method than ether narcosis.

Anesthetics, Inhalation↗

PubMed1986

Low density lipoprotein degradation by secretory granules of rat mast cells. Sequential degradation of apolipoprotein B by granule chymase and carboxypeptidase A.

The secretory granules of rat serosal mast cells are able efficiently to degrade the apolipoprotein B component of low density lipoproteins (LDL) Kokkonen, J. O., and Kovanen, P. T. (1985) J. Biol. Chem. 260, 14756-14763). The granules are known to contain two neutral proteases with complementary specificities: a chymotrypsin-like endopeptidase called chymase, and an exopeptidase, the granule carboxypeptidase A. The role of this enzyme pair in the proteolytic degradation of LDL was studied with the aid of specific enzyme inhibitors. Incubation of LDL with intact granules (both enzymes active) led to the formation of numerous low molecular weight peptides and the liberation of free amino acids, most of which (95%) were aromatic (Phe, Tyr, Trp) or branched-chain aliphatic (Leu, Ile, Val). Selective inhibition of granule carboxypeptidase A (leaving chymase active) blocked the liberation of free amino acids, but left the formation of peptides uninhibited. On the other hand, selective inhibition of granule chymase (leaving carboxypeptidase A active) totally abolished the proteolytic degradation of LDL. The results are consistent with a model according to which the proteolytic degradation of LDL by mast cell granules results from coordinated action of the two granule-bound enzymes, whereby the chymase first cleaves peptides from the apolipoprotein B of LDL, and thereafter the carboxypeptidase A cleaves amino acids from the peptides formed.

Amino Acids↗

PubMed1993

Studies of 5-fluorodeoxyuridine 5'-monophosphate binding to carboxypeptidase A-inactivated thymidylate synthase from Lactobacillus casei.

The binding of 5-fluorodeoxyuridylate (FdUMP) to carboxypeptidase-inactivated thymidylate synthase obtained from methotrexate-resistant Lactobacillus casei was investigated using [3H]FdUMP in a trichloroacetic acid precipitation assay and by 19F nuclear magnetic resonance spectroscopy. The cleavage of 1 valine residue from the carboxyl terminus of one of the identical subunits of the enzyme dimer correlates with complete loss of thymidylate synthesis (Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). We have further investigated the phenomenon of carboxypeptidase A-dependent inactivation of thymidylate synthase by employing immobilized carboxypeptidase A in order to facilitate the isolation and characterization of the inactivated enzyme. The time course of carboxypeptidase treatment of thymidylate synthase has been profiled by the spectrophotometric assay, tritium release assay, trichloroacetic acid precipitation assay (covalent adduct analysis), 19F nuclear magnetic resonance spectroscopy, and amino acid analysis. The techniques utilized in this study yielded results which showed that the completely inactivated enzyme (failure to catalyze thymidylate formation) continued to catalyze both covalent FdUMP-enzyme interactions and the formation of the covalent inhibitory ternary complex with the cofactor, 5,1O-methylenetetrahydrofolate, although to a reduced extent, thus effectively uncoupling these processes from thymidylate synthesis activity.

Binding Sites↗

PubMed1987

Maturation of vacuolar (lysosomal) enzymes in yeast: proteinase yscA and proteinase yscB are catalysts of the processing and activation event of carboxypeptidase yscY.

Studies were performed to unravel the activation and maturation mechanism of vacuolar (lysosomal) proteinases in Saccharomyces cerevisiae. In vivo and in vitro studies show that proteinase yscA and proteinase yscB are involved in the activation and processing event of pro-carboxypeptidase yscY. Processing and activation of pro-carboxypeptidase yscY by proteinase yscA depends on an additional factor contained in the vacuolar fraction. Comparable activation can be mimicked by sodium polyphosphate. Optimum pH for processing by this proteinase yscA-triggered event is 5. The proteinase yscA-triggered maturation process of pro-carboxypeptidase yscY leads to an intermediate mol. wt form of the enzyme which is, however, fully active. Proteinase yscB transfers the intermediate mol. wt form of the original precursor to the apparently authentic, mature and active carboxypeptidase yscY. An activation and maturation scheme is devised.

Aspartic Acid Endopeptidases↗

PubMed1994

A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the alpha/beta hydrolase fold, acts as a pH-dependent protein-stabilizing element.

Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.

Amino Acid Sequence↗

PubMed1992

Human serum carboxypeptidase U: a new kininase?

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its instability at room temperature and at 37 degrees C, we named it "unstable carboxypeptidase" (carboxypeptidase U, CPU). The enzymatically active subunit of carboxypeptidase U was purified and exhibits a single band at 53 kDa on SDS-PAGE.

Carboxypeptidases↗

PubMed1993

The carbohydrate moiety of the acid carboxypeptidase from Aspergillus saitoi.

Acid carboxypeptidase from Aspergillus saitoi is a glycoprotein that contains both N- and O-linked sugar chains. The N-glycanase released high-mannose type oligosaccharides that were separated into eight components on HPLC. One, which had a unique structure of Man11GlcNAc2, was characterized. Mild alkali treatment of the carboxypeptidase, under conditions that effect beta-elimination, yielded D-mannose. Deglycosylation of the carboxypeptidase with endo-beta-N-acetylglucosaminidase and alpha-mannosidase effected the reduction of the molecular mass from 72 kDa to 60 kDa. Partial changes of CD spectra of the native and the deglycosylated enzymes indicate that some conformational changes on the peptide of the enzyme occurred after deglycosylation. Other enzymatic properties, such as catalytic activity, pH, and thermal stability and resistivity to protease digestion, did not appear to change. Tunicamycin halted secretion of the carboxypeptidase extracellularly.

Aspergillus↗

PubMed1988

Carboxypeptidase S-1 from Penicillium janthinellum: enzymatic properties in hydrolysis and aminolysis reactions.

Carboxypeptidase S-1 from Penicillium janthinellum has been isolated by affinity chromatography and characterized. The enzyme activity is unusually stable in organic solvents, e.g. 80% methanol. The hydrolysis of peptide substrates is apparently dependent on three ionizable groups. One group, with pKa of 4.0-4.5, is a catalytically essential residue in its deprotonated form, and another group with a pKa of 6.5-7.0 functions in its protonated form, apparently as the binding site for the C-terminal carboxylate group of peptide substrates. The third group, with a pKa of 5.0-5.5, appears to be a carboxylic acid group located at the S1 binding site. Deprotonation of this group to form a negatively charged carboxylate group has an adverse effect on the hydrolysis of substrates with hydrophobic amino acid residues at the P1 position and a beneficial effect on the hydrolysis of substrates with the positively charged arginyl or lysyl residues at this position. The substrate preference of the enzyme is consequently pH dependent. At pH 6.0 (the optimum for ester hydrolysis) the enzyme is essentially specific for Bz-X-OMe substrates where X = Arg and Lys. Using amino acids and amino acid amides as nucleophiles carboxypeptidase S-1 efficiently catalyses the formation of peptide bonds by aminolysis of peptides (transpeptidation reactions) and peptide esters provided that the substrate contains a basic amino acid residue at the P1 position, e.g. Bz-Arg-OBu and Bz-Arg-Leu-OH. With several nucleophiles the fractions of aminolysis exceed those previously reported in similar studies with carboxypeptidase Y and malt carboxypeptidase II.

Carboxypeptidases↗

PubMed1996

Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini.

Amino Acid Sequence↗

PubMed2004

Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus.

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50 degrees C for MpiCP-1, and 3.5 and 50 degrees C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37 degrees C for 1 h, and up to 55 degrees C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50 degrees C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-L: -tyrosyl-L: -glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The Km, Vmax, Kcat and Kcat/Km values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37 degrees C were 1.33 mM, 1.49 mM min(-1), 723 s(-1) and 545 mM(-1) s(-1), and those of MpiCP-2 at pH 3.5 and 37 degrees C were 1.55 mM, 1.54 mM min(-1), 2,039 s(-1) and 1,318 mM(-1) s(-1), respectively.

Carboxypeptidases↗

PubMed1975

Purification of a carboxypeptidase B-like enzyme from the starfish Dermasterias imbricata.

A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters of lysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.

Animals↗