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PubMedDate not supplied

[Effect of testosterone on carboxypeptidase H activity in the murine hypothalamus and hypophysis].

Effects of single intraperitoneal administration of testosterone propionate (3 or 30 mg/kg body weight) on activity of carboxypeptidase H in pituitary body and hypothalamus of female white mouse were studied. It was found that testosterone propionate treatment (3 and 30 mg/kg body weight) increased the carboxypeptidase H activity in pituitary through 0.5 hour and it decreased one in 24 hours after treatment. The carboxypeptidase H activity in hypothalamus was lower as compared with control animals in 24 hours after testosterone propionate treatment in the dose 3 mg/kg body weight. However, the carboxypeptidase H activity in hypothalamus was lower in 0.5 and 24 hours and it was higher in 4 h after testosterone propionate treatment in the dose 30 mg/kg body weight as compared with the control. These data suggest that testosterone affects the carboxypeptidase H activity by changing the level of enzyme gene expression.

Animals↗

PubMed1990

Human carboxypeptidase A identifies a BglII RFLP and maps to 7q31-qter.

A genomic clone for human carboxypeptidase has been isolated with a probe for rat CPA1 cDNA. A 1.7-kb HindIII/EcoRI fragment from the 3' flanking region of human carboxypeptidase detects a DNA polymorphism with BglIII. Multipoint linkage analysis with an established map of chromosome 7 markers shows that the most likely location of carboxypeptidase is at 7q31-qter, between D7S87 and D7S93. All other placements can be excluded with odds greater than 100:1. These and other markers confirm that carboxypeptidase lies distal to the locus for cystic fibrosis, at a distance of approximately 12 centimorgans. The regions containing identity to the rat gene were sequenced and shown to be 82% identical to exons 9 and 10 of the rat gene. The presence of a codon for isoleucine at the residues corresponding to codon 255 of rat CPA1 cDNA strongly suggests that the A form of human carboxypeptidase has been isolated.

Bacterial Proteins↗

PubMed1988

Characterization of the carboxypeptidase N secreted by Hep G2 cells.

Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.

Animals↗

PubMed1978

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.

Acylation↗

PubMed1985

Radioimmunoassay of active pancreatic enzymes in sera from patients with acute pancreatitis. I. Active carboxypeptidase B.

Previous studies have suggested that measurement of active enzymes in relation to proenzymes in serum of patients with pancreatitis may reflect the degree of zymogen activation in the gland. Here we describe the first single-tube assay for an active form of a pancreatic enzyme that is ordinarily synthesized as a proenzyme. Human procarboxypeptidase B, which we purified to near homogeneity, is approximately 13 000 Da larger than the active enzyme (EC 3.4.17.2). Antibodies specific for active carboxypeptidase B were obtained by affinity chromatography of anti-carboxypeptidase B antisera on a gel containing procarboxypeptidase B, then used to develop a single-tube radioimmunoassay for measuring active carboxypeptidase B in serum. Using this assay, we were able to detect, for the first time, active carboxypeptidase B in sera from patients with acute pancreatitis. Preliminary data show a correlation between the serum concentrations of active carboxypeptidase B and those of active trypsin complexed with serum inhibitors, but no correlation with serum amylase values.

Acute Disease↗

PubMed1989

Identification of proline-specific carboxypeptidase localized to brush border membrane of rat small intestine and its possible role in protein digestion.

A proline-specific carboxypeptidase (carboxypeptidase P, EC 3.4.12.-) was identified and partially characterized in the brush border membrane fraction of rat intestinal enterocytes and shown to be distinct from pancreatic proteases. The carboxypeptidase activity of isolated brush border membranes, with Z-Gly-Pro-Leu as substrate, was 43 nmol/min/mg protein representing a 16-fold purification when compared with mucosal cell homogenates. Activity was maximal in the middle region of the small intestine, and villus cells had twice the activity of crypt cells. Carboxypeptidase activity was maximal at pH 7.0, was stimulated by divalent cations, and was inhibited by metal chelating agents, suggesting that it is a metalloenzyme. The enzyme had the highest activity with synthetic peptides containing proline penultimate to the carboxy terminus. In vivo patterns of hydrolysis and absorption of amino acids from Z-Pro-Trp were examined using an intestinal perfusion technique. These studies indicate that brush border membrane carboxypeptidase may play an important role in the digestion of proline-containing peptides and proteins.

Animals↗

PubMed2001

Carboxypeptidases from A to z: implications in embryonic development and Wnt binding.

Carboxypeptidases perform many diverse functions in the body. The well-studied pancreatic enzymes (carboxypeptidases A1, A2 and B) are involved in the digestion of food, whereas a related enzyme (mast-cell carboxypeptidase A) functions in the degradation of other proteins. Several members of the metallocarboxypeptidase gene family (carboxypeptidases D, E, M and N) are more selective enzymes and are thought to play a role in the processing of intercellular peptide messengers. Three other members of the metallocarboxypeptidase gene family do not appear to encode active enzymes; these members have been designated CPX-1, CPX-2 and AEBP1/ACLP. In this review, we focus on the recently discovered carboxypeptidase Z (CPZ). This enzyme removes C-terminal Arg residues from synthetic substrates, as do many of the other members of the gene family. However, CPZ differs from the other enzymes in that CPZ is enriched in the extracellular matrix and is broadly distributed during early embryogenesis. In addition to containing a metallocarboxypeptidase domain, CPZ also contains a Cys-rich domain that has homology to Wnt-binding proteins; Wnts are important signaling molecules during development. Although the exact function of CPZ is not yet known, it is likely that this protein plays a role in development by one of several possible mechanisms.

Amino Acid Sequence↗

PubMed2003

DNA polymorphism and mutations in CPN1, including the genomic basis of carboxypeptidase N deficiency.

Carboxypeptidase N (EC 3.4.17.3) regulates the activity of peptides such as kinins and anaphylatoxins. Although deficiency of carboxypeptidase N (MIM 212070) produces a severe allergic syndrome, no human mutations have ever been described. Therefore, using archival genomic DNA from a subject with documented carboxypeptidase N deficiency, we sequenced CPN1 (MIM 603103), which encodes the catalytic subunit of carboxypeptidase N. In the genomic DNA of the proband, we discovered three CPN1 variants: (1) 385fsInsG, a frameshift mutation in exon 1 due to a single G insertion at nucleotide 385; (2) 746G>A single-nucleotide polymorphism (SNP), a missense mutation in exon 3 that predicted substitution of aspartic acid for the wild-type conserved glycine at amino acid 178 (G178D); and (3) IVS1 +6C>T, an SNP in intron 1. Among 128 normal Caucasians, the 385fsInsG mutation was absent and the G178D mutation had a frequency of 0.0078, suggesting that these were rare molecular events that likely contributed to the carboxypeptidase N deficiency phenotype. The frequency of the IVS1 +6C>T polymorphism was 0.051. The reagents described here provide tools for further study of association with clinical and biochemical phenotypes related to allergy and immunity.

Aged↗

PubMed1988

Comparison of a carboxypeptidase E-like enzyme in human, bovine, mouse, Xenopus, shark and Aplysia neural tissue.

Several diverse species contain an enzyme with many properties in common with those of bovine carboxypeptidase E (CPE), a neuropeptide processing carboxypeptidase B-like enzyme. This enzyme has been designated EC 3.4.17.10, and is also known as enkephalin convertase and carboxypeptidase H. All tissues that are known to contain bioactive peptides also contain CPE-like enzymatic activity. In Xenopus laevis, enzyme activity is highest in the brain and pituitary, lower in the skin, and undetectable in liver and gut. In Aplysia californica, enzyme activity is highest in the atrial gland, but is also present in moderate amounts in the various neural tissue. CPE extracted from human, bovine, mouse, Xenopus, shark, and Aplysia neural tissue is substantially purified using substrate affinity chromatography and concanavalin A sepharose columns. The partially purified enzyme from all species examined possess very similar enzymatic properties. These properties include a pH optimum of 5.6, a stimulation by cobalt chloride, and an inhibition by chelating agents (1,10-phenanthroline). Arginine-derived active site-directed inhibitors show similar inhibition constants (Ki's) towards enzyme from the various species, whereas lysine-derived inhibitors are substantially less potent towards the Aplysia carboxypeptidase than towards enzyme isolated from the other species. The similar properties of the carboxypeptidase isolated from the various species suggests that a CPE-like is involved in the biosynthesis of many peptide neurotransmitters and hormones in a wide range of organisms.

Animals↗

PubMed1991

Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase T. A metalloenzyme endowed with dual substrate specificity.

A gene coding for an extracellular Zn-carboxypeptidase of Thermoactinomyces vulgaris has been cloned and sequenced (EMBL X56901). This enzyme named carboxypeptidase T reveals simultaneously both types of substrate specificity characteristic of mammalian carboxypeptidases A and B. The carboxypeptidase T gene is primarily expressed in E. coli as a non-active preproenzyme with an additional 98 amino acid residues at the N-terminus. Primary structure alignment of mature carboxypeptidase T and mammalian metallocarboxypeptidases demonstrated 25-30% overall identity but a full preservation of presumed catalytically important residues. These observations imply a basic uniformity of the general catalytic mechanism for enzymes of that class produced by evolutionarily remote organisms.

Amino Acid Sequence↗

PubMed1995

The primary structure of carboxypeptidase S3 from Penicillium janthinellum IBT 3991.

The complete amino acid sequence of penicillopeptidase S3, a serine carboxypeptidase isolated from Penicillium janthinellum IBT 3991, has been determined. The enzyme consists of 481 amino acids arranged in a single polypeptide chain. Six glycosylation sites were established in positions 41, 218, 256, 326, 384 and 392. The molecule contains six cysteinyl residues among which disulfide bridges was established between Cys-71-Cys-333 and Cys-233-Cys-289. Carboxypeptidase S3 is homologous to carboxypeptidase PEPF (or carboxypeptidase I) from Aspergillus niger (67% identical positions). It is proposed that these enzymes form a separate sub-family among the serine carboxypeptidases.

Amino Acid Sequence↗

PubMed1986

Pancreatic carboxypeptidase hydrolysis of bile acid-amino conjugates: selective resistance of glycine and taurine amidates.

To find a possible explanation for the selective hepatic conjugation of bile acids with glycine or taurine, the N-acyl amidates of cholic acid and a number of amino acids and amino acid analogues were synthesized, and their susceptibility to hydrolysis by pancreatic juice, gastric juice, serum, or small intestinal mucosal enzymes was measured. Deconjugation by pure carboxypeptidase A and B was also examined, and hydrolysis by these tissue fluids and enzymes was compared with that mediated by a bacterial cholylglycine hydrolase. Human pancreatic juice efficiently hydrolyzed cholyl conjugates of all neutral-L-amino acids (cholyl-L-alanine, cholyl-L-valine, cholyl-L-leucine, and cholyl-L-tyrosine), except cholylglycine. The net hourly rate of hydrolysis (in micromoles per milligram protein per hour) increased when the terminal residue was aromatic or branched aliphatic, and appeared to be specific for L-alpha-amino acids as cholyl-beta-alanine and cholyl-D-valine were not cleaved. From cholyl glycylglycine, only the terminal glycine was efficiently removed. Cholyltaurine and cholyl conjugates with the methyl and propyl analogues of taurine were resistant to hydrolysis. Two basic amino acid conjugates (cholyl-L-lysine and cholyl-L-arginine) were cleaved, whereas conjugates of acidic amino acids (cholyl-aspartate and cholyl-cysteate) were not cleaved. Studies using pure enzymes showed that bovine carboxypeptidase A hydrolyzed the cholyl conjugates of the neutral L-alpha-amino acids with similar specificity as observed for the human pancreatic juice, whereas bovine carboxypeptidase B cleaved the basic amino acid conjugates. Cholyl-L-lysine and cholyl-L-arginine were also cleaved by serum and plasma, which are known to possess carboxypeptidase activity. Cholyl conjugates were not cleaved by gastric juice, by trypsin, or by homogenates of rat small intestinal mucosa. In contrast, all cholyl conjugates were cleaved by a bacterial cholylglycine hydrolase. These experiments indicate that glycine and taurine amidates of cholic acid differ from a number of other conjugates with neutral and basic amino acid in being resistant to hydrolysis by pancreatic and plasma carboxypeptidases.(ABSTRACT TRUNCATED AT 400 WORDS)

Amidohydrolases↗

PubMed1997

Identification of a membrane-bound carboxypeptidase as the mammalian homolog of duck gp180, a hepatitis B virus-binding protein.

A unique membrane-bound carboxypeptidase was discovered and characterized in membrane fractions of human skin fibroblasts and the mouse monocyte-macrophage cell line J774A.1 and was partially purified from human placenta. Enzymatic characterization identified it as a new member of the regulatory B-type metallocarboxypeptidases, different from carboxypeptidases B, E, M, N and U. It is, however, similar to the newly described bovine carboxypeptidase D, suggested to be a homolog of duck gp180, a 180 kDa hepatitis B virus-binding protein. To prove this, a partial cDNA encoding a 20 kDa fragment of the human homolog of duck gp180 was expressed in bacteria and the recombinant protein was purified. Antibodies raised to the protein immunoprecipitated 94% or 72% of the low pH carboxypeptidase activity in human skin fibroblasts or J774A.1 cells and gave a 175 kDa protein band in Western blots. Thus, carboxypeptidase D is the mammalian homolog of duck gp180 and is distributed in a variety of different cell types.

Animals↗

PubMed1991

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules.

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.

Amino Acid Sequence↗

PubMed1989

Carboxypeptidase-mediated release of methotrexate from methotrexate alpha-peptides.

Methotrexate (MTX) alpha-peptides containing representative neutral (alanine), acidic (aspartic acid), and basic (arginine) amino acids were synthesized by a regiospecific route. Purity and authenticity of MTX-Ala, MTX-Asp, and MTX-Arg were established by TLC, HPLC, elemental analysis, and NMR and absorbance spectra. These peptides were hydrolyzed by carboxypeptidases to yield MTX and the amino acids. Reactions were monitored by using a ninhydrin assay for the amino acids and HPLC and spectrophotometric assays for MTX. Pancreatic carboxypeptidase A (CP-A) hydrolyzed MTX-Ala and, at a much slower rate, MTX-Asp and MTX-Arg. MTX-Ala was also a substrate for pancreatic carboxypeptidase B (CP-B); marginal activity was observed with this enzyme and MTX-Arg. Human serum hydrolyzed only MTX-Arg; biphasic inhibition of this activity by 2-(mercaptomethyl)-3-(guanidinoethyl)thiopropionate was consistent with the known presence of two types of endogenous carboxypeptidase (CP-N). Cytotoxicity of the MTX peptides toward L1210 cells in culture was enhanced considerably in the presence of the appropriate carboxypeptidases. MTX-Ala was much less toxic than MTX (ID50 values of 2.0 X 10(-6) M and 2.4 x 10(-8) M, respectively), but in the presence of CP-A the ID50 of the peptide improved to 8.5 X 10(-8) M. Similar results were obtained with MTX-Asp/CP-A and MTX-Ala/CP-B combinations. MTX-Arg showed good cytotoxicity (ID50 of 5.0 X 10(-8) M), due to CP-N activity in the fetal bovine serum of the culture medium; inclusion of CP-B lowered the ID50 to that of MTX. Possible clinical uses of MTX peptides are discussed.

Amino Acids↗

PubMed1998

A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins.

A 25-kDa inhibitor of the vacuolar enzyme carboxypeptidase Y from Saccharomyces cerevisiae has been characterized. The inhibitor, Ic, binds tightly with an apparent Ki of 0.1 nM. Consistent with a cytoplasmic localization, Ic is soluble and contains no sequences which could serve as potential signals for transport into the endoplasmic reticulum. Surprisingly, Ic is encoded by TFS1, which has previously been isolated as a high-copy suppressor of cdc25-1. CDC25 encodes the putative GTP exchange factor for Ras1p/Ras2p in yeast. In an attempt to rationalize this finding, we looked for a physiological relationship by deleting or overexpressing the gene for carboxypeptidase Y in a cdc25-1 strain. However, this did not change the phenotype of this mutant strain. Ic is the first member of a new family of protease inhibitors. The inhibitor is not hydrolyzed on binding to CPY. It has fairly high degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several higher eukaryotes, including man. These proteins are highly abundant in some tissues (e.g., brain) and have in general been found to bind lipids. Considering their homology to Ic, it is tempting to speculate that they may also be inhibitors of serine carboxypeptidases.

Amino Acid Sequence↗

PubMed1983

The N alpha-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity.

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA--Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.

Animals↗

PubMed1984

Human 'creatine kinase conversion factor' identified as a carboxypeptidase.

The effect of partially purified 'creatine kinase conversion factor' on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.

Animals↗