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PubMed1994

Identification of the C-terminal amino acid amides by carboxypeptidase Y digestion and fast atom bombardment mass spectrometry.

The combination method of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry is described for the identification of C-terminal amino acid amides in peptides. Carboxypeptidase Y has amidase activity as well as exopeptidase activity in the same digestion buffer condition. Based on this concept, we develop a new technique which can definitively and easily identify the C-terminal amino acid amides. This method obviates the need for several complicated steps occurring in previous methods, but improves sensitivity, and enables exact identification of the amino acid amide by the difference of molecular mass. Analyses of carboxypeptidase Y digested peptides, not liberated free amino acid amides, were carried out by fast atom bombardment mass spectrometry. The use of truncated peptides by fast atom bombardment mass spectrometry in C-terminal amino acid amide determination gives several advantages over analyses of the liberated amino acid amides. The C-terminal amino acid amides of Allantostatin I (Leu-NH2), alpha-Melanocyte Stimulating Hormone (Val-NH2), and Ranatensin (Met-NH2) are unequivocally determined at a level of 0.90-2.3 nmol per peptide. This approach is based on entirely different principles than the previous approaches.

Amides↗

PubMed1994

Recognition of C-terminal amide groups by (serine) carboxypeptidase Y investigated by site-directed mutagenesis.

Serine carboxypeptidases have the ability to hydrolyze peptides as well as peptide amides. Previously, it has been demonstrated that Asn51 and Glu145 (in the protonated form) each donate a hydrogen bond to the alpha-carboxylate of peptide substrate. It is here demonstrated by characterization of carboxypeptidase Y derivatives, mutationally altered at positions 51 and 145, that the same groups are involved in the interaction with the C-terminal carboxyamide group of peptide amides. Asn51 donates a hydrogen bond to the C = O group of the substrate, and Glu145 (in the charged form) accepts one from the NH2 group of the substrate. Thus, the ionic state of Glu145 is different when peptides are hydrolyzed as compared with when peptide amides are hydrolyzed. This explains why Km for the hydrolysis of peptides increases with pH, whereas it remains constant for peptide amides. As a consequence, kcat/Km for the hydrolysis of peptide amides is higher than for the hydrolysis of peptides at pH > 8. At physiological pH, peptides and peptide amides are hydrolyzed with rates of the same order of magnitude; this is in accordance with reports describing that serine carboxypeptidases are involved in the degradation of biologically active peptide amides.

Amidohydrolases↗

PubMed1993

Sequencing and cloning of human prolylcarboxypeptidase (angiotensinase C). Similarity to both serine carboxypeptidase and prolylendopeptidase families.

Prolylcarboxypeptidase, a lysosomal serine carboxypeptidase, cleaves COOH-terminal amino acids linked to proline, as in angiotensin II and III and [des-Arg9] bradykinin. About 25% of the enzyme protein was sequenced, and the complete sequence was deduced from its human kidney cDNA. The cDNA insert contained an open reading frame of 1488 base pairs coding for a protein of 496 residues. The authentic NH2-terminal sequence matched the deduced protein sequence starting with residue 46, suggesting the presence of both a signal and propeptide. The mature enzyme (451 residues) has a calculated M(r) = 51,043, whereas the M(r) of the purified glycoprotein is 58,000, indicating 12% carbohydrate. The overall sequence identity with serine peptidases is low (10-18%), but sequences around residues of the putative catalytic triad (Ser134, Asp333, His411) are similar (30-67%) to both the serine carboxypeptidases (e.g. deamidase or lysosomal protective protein, yeast carboxypeptidase Y, and KEX1 gene product) and the prolylendopeptidase family. Thus, prolylcarboxypeptidase links these two families, suggesting an evolutionary relationship. It is inhibited (Ki = 2.6 x 10(-7) M) by benzyloxycarbonyl-Pro-prolinal, a specific inhibitor of prolylendopeptidase, another angiotensin metabolizing enzyme. Prolylcarboxypeptidase contains serine or threonine residues repeated as the 26th residue 7 out of 9 times, with identical or similar amino acids in other positions in the repeats. The KEX1 gene product contains a similar motif, with serine or threonine as every 27th residue. The importance of prolylcarboxypeptidase is strongly suggested by its presence in various organs and cells and by the substrates it cleaves.

Amino Acid Sequence↗

PubMedDate not supplied

[Properties of basic (splitting off arginine and lysine residues) carboxypeptidases and their role in the functioning of biologically active peptides].

Properties of carboxypeptidases that cleaved arginine and lysine from C-terminus of peptides are considered. Their role in processing, modulation and inactivation of active peptides are discussed. Mechanisms of regulation of carboxypeptidase H activity are considered.

Arginine↗

PubMed1997

Gem-dialkyl succinic acids: a novel class of inhibitors for carboxypeptidases.

gem-Dimethylsuccinic acid and its higher homolog, 2-methyl-2-ethylsuccinic acid (MESA) are highly potent inhibitors of both carboxypeptidase A (CPA) and B. The inhibition constant of MESA for CPA (0.11 microM for the racemic mixture) is remarkable considering the relatively simple structure of the compound. The molecular feature which is crucial for high affinity binding to both carboxypeptidases appears to be the nonpolar gem-dialkyl locus. The structure of the complex between MESA and CPA has been determined by X-ray crystallography to 2.0 A resolution and shows the R enantiomer of the inhibitor to be bound in a generally substrate-like manner. The carboxymethyl group is coordinated to the Zn ion in the active site, and the gem-dialkyl locus corresponds in position to the alpha-carbon of the C-terminal amino acid in a peptide substrate. The methyl group of the inhibitor occupies a cavity in the enzyme which is apparently not filled upon substrate-binding. We postulate that this cavity (the alpha-methyl hole) is designed to allow the proximal Glu-270 residue to undergo a critical movement during catalysis. The hydrophobic nature of the above cavity may play a role in modulating the reactivity of this residue. These results suggest that similar cenophilic(empty-loving) inhibitors may be found for other enzymes.

Animals↗

PubMed2000

Molecular cloning and partial characterization of rat procarboxypeptidase R and carboxypeptidase N.

Carboxypeptidase R (EC 3.4.17.20) (CPR) and carboxypeptidase N (EC 3.4.17.3) (CPN) cleave carboxy-terminal arginine or lysine residues from biologically active peptides such as kinins or anaphylatoxins in the circulation thereby regulating their activities. Although CPN is present in a stable active form in plasma, CPR is generated from proCPR, a plasma zymogen, by proteolytic enzymes such as thrombin, thrombin-thrombomodulin complex and plasmin. We have isolated rat proCPR and CPN cDNA clones which can induce enzymatic activities in culture supernatants of the transfected cells. mRNA of proCPR was detected only in rat liver by Northern hybridization and showed hepatocyte-specific expression. Expression of proCPR mRNA was enhanced following LPS injection, indicating that proCPR production is increased under inflammatory conditions.

Animals↗

PubMed2002

Heat stability of carboxypeptidase R of experimental animals.

Carboxypeptidase N (CPN) and carboxypeptidase R (CPR) are present in fresh serum, and cleave C-terminal arginine or lysine residues from bioactive peptides such as anaphylatoxins and kinins resulting in regulation of peptide activity. Although CPN is present in the active form in plasma, CPR is generated from proCPR by trypsin-like enzymes such as thrombin. CPR regulates not only inflammatory peptides but also restricts fibrinolysis. To elucidate the complex role of CPN and CPR in vivo, studies in animal models will be essential. CPR of guinea pig, rat and rabbit decayed at 37 C rapidly as in the case of human CPR. However, at 25 C, CPR of those species decayed to some extent, although human serum CPR did not decay within 60 min. In the presence of thrombin inhibitor, CPR in the sera of animals tested decayed more rapidly than CPR in serum without thrombin inhibitor suggesting that additional generation of CPR may have been prevented during decay evaluation. However, human serum CPR decayed more rapidly in the absence of thrombin inhibitor indicating that thrombin may accelerate the decay in human serum.

Animals↗

PubMedDate not supplied

[Substrates for enkephalin-forming carboxypeptidase in the rat brain and adrenals].

Dansyl-Phe-Ala-Arg and (D-Ala2, Leu5) enkephalin-Arg6 (dalargine) were studied as possible substrates for enkephalin-forming carboxypeptidase (EFC) and carboxypeptidase B (CPB) in crude tissue homogenates. Activities of these enzymes in rat cerebellum, pituitary and adrenal glands were determined using the dansyl substrate. Kinetic characteristics of CPB and EFC were evaluated for dansyl substrate and dalargine. Affinity of EFC to dalargine was 10-fold higher as compared with CPB. Dalargine may be used as a specific substrate for study of EFC in tissue preparations.

Adrenal Glands↗

PubMed2006

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli.

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated.

Dipeptidases↗

PubMed1983

A dipeptidyl carboxypeptidase in brain synaptic membranes active in the metabolism of enkephalin containing peptides.

A dipeptidyl carboxypeptidase activity has been localized in synaptic plasma membranes which have been prepared from isolated rat brain cortical synaptosomes. The specificity of this proteolytic activity towards various synthetic and biological active peptides is compared to the peptidase activities of intact synaptosomes. In contrast to the synaptosomal peptidases which are capable of cleaving all peptide bonds of Met-enkephalin-Arg6-Phe7 the peptidase activity associated with the synaptic plasma membrane exclusively hydrolyses a dipeptide from the carboxyl terminus of all hepta- and hexapeptides tested. The fact that this dipeptidyl carboxypeptidase does not cleave the Gly3-Phe4 peptide bond of Met-enkephalin suggests that this enzyme is different from "enkephalinase". The synaptic membrane dipeptidyl carboxypeptidase is inhibited by metal chelating agents and thiols but is not affected by compounds known to inhibit serine proteases, thermolysin and "enkephalinase".

Amino Acid Sequence↗

PubMed1996

Membrane anchoring and release of carboxypeptidase M: implications for extracellular hydrolysis of peptide hormones.

Carboxypeptidase M (CPM) was discovered as a membrane-bound B-type carboxypeptidase which is widely distributed in a variety of tissues and cells. The amino acid sequence of CPM indicated that the C-terminal hydrophobic region might be a signal for membrane attachment via a glycosylphosphatidylinositol (GPI) anchor. This was demonstrated by [3H)ethanolamine labeling of Madin Darby canine kidney (MDCK) cells which resulted in labeling of the membrane anchor of CPM as shown by immunoprecipitation, polyacrylamide gel electrophoresis and autoradiography. Trypsin released CPM from the membrane, resulting in removal of the radiolabeled ethanolamine. Carboxypeptidase activity was spontaneously and continuously released from MDCK cells into the medium. The released enzyme is a soluble form of CPM as shown by Triton X-114 partitioning, immunoprecipitation, Western blotting, inhibition studies and its neutral pH optimum. CPM was also found in soluble form in biological fluids such as urine and amniotic fluid where it is the primary enzyme that hydrolyzes epidermal growth factor (EGF), producing des-Arg53-EGF. These data indicate that CPM is involved in peptide metabolism on both the cell surface and in extracellular fluids.

Amino Acid Sequence↗

PubMed2004

Secondary binding site of the potato carboxypeptidase inhibitor. Contribution to its structure, folding, and biological properties.

The contribution of each residue of the potato carboxypeptidase inhibitor (PCI) secondary binding site to the overall properties of this protein has been examined using alanine-scanning mutagenesis. Structural and enzymatic studies, performed on a series of PCI mutants, demonstrate that the proper positioning of the primary site for efficient binding and inhibition of carboxypeptidase A is significantly dependent on such a secondary contact region. The aromatic residues in this region play a key role in the stabilization of the PCI-enzyme complex, whereas polar residues contribute little to this task. A comparative study of the oxidative folding of these PCI mutants has been carried out using the disulfide quenching approach. The data, together with the structural characterization of some of these mutants, clearly indicate that noncovalent forces drive the refolding of this small disulfide-rich protein at the reshuffling stage, the rate-limiting step of the process. Moreover, it reveals that by introducing new noncovalent intramolecular contacts in PCI, we may create more stable variants, which also show improved folding efficiency. Taken together, the collected results clarify the folding determinants of the primary and secondary binding sites of PCI and their contribution to the inhibition of the carboxypeptidase, providing clues about PCI evolution and knowledge for its biotechnological redesign.

Binding Sites↗

PubMed1978

Escherichia coli mutants defective in dipeptidyl carboxypeptidase.

Two independent mutants of Escherichia coli deficient in dipeptidyl carboxypeptidase activity (Dep-) were isolated after mutagenesis with ethyl methanesulfonate. Mating experiments and introduction of specific episomes indicated that the responsible gene was located at approximately 27--31 min on the E. coli chromosome. The Dep- mutants differed from the parental strain in their inability to grow with N-acetylalanylalanylalanine as the sole nitrogen source. Revertants selected for growth on this substrate of the enzyme were found to have reacquired the activity. Enzyme activity was highly sensitive to inhibition by 1-(D-3-mercapto-2-methylpropanoyl)-L-proline (SQ 14225), a potent inhibitor of mammalian dipeptidyl carboxypeptidase (angiotensin-converting enzyme, peptidyl dipeptidase, EC 3.4.15.1). This compound also reduced the rate of growth of the wild type with N-acetylalanylalanylalanine but not with ammonium sulfate. A fraction of the enzyme was released into the medium by osmotic shock, indicating that its presence in the periplasmic space may account for growth with N-acetylated peptides that cannot be taken up by E. coli. In addition to providing information about the specific role of this exopeptidase in E. coli, the Dep- mutants may prove useful for delineating the regulation and cellular function of dipeptidyl carboxypeptidases in higher organisms.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1980

Variations through the day of hepatic and muscular cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D; EC 3.4.23.5) activities and free amino acids of blood in rats: influence of feeding schedule.

1. Growing rats were fed either ad lib. or with six (equal) meals offered every 4 h (from 10.00 hours). Rats of each group were killed at intervals of 4 h beginning at 11.00 hours. Activities of cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D EC 3.4.23.5) were measured in liver and muscle homogenates and free amino acids in blood were determined. 2. In the rats fed ad lib. activities of carboxypeptidase A and endopeptidase D in liver and muscle showed significant variation, with maximum activity in the light period. In general, meal-feeding only caused minor differences in cathepsin activities; although significant differences occurred for carboxypeptidase A. For the later enzyme a peak in activity occurred in the dark as well as in the light period. 3. Irrespective of the feeding schedule, the lower concentration of free essential amino acids of blood occurred generally during the night period. With the controlled-feeding schedule there is an increase of essential amino acids and a slight decrease of non-essentail amino acids of blood.

Amino Acids↗

PubMed1996

Isolation and Identification of Ripening-Related Tomato Fruit Carboxypeptidase.

Tomato (Lycopersicon esculentum) fruit carboxypeptidase active on N-carbobenzoxy Z-L-phenylalanine-L-alanine was found to constitute a family of isoforms whose abundance changed differentially during ripening. A specific polyclonal antibody against the fruit carboxypeptidase was raised in rabbits and used to purify and identify the protein. The data from immunoaffinity chromatography, immunoinhibition studies, immunoprecipitation of the in vivo- and in vitro-labeled proteins, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of native isoforms strongly suggest that the fruit carboxypeptidases are monomers or oligomers of 68- and/or 43-kD subunits.

Journal Article↗

PubMed1978

Development and Localization of Carboxypeptidase Activity in Embryo-less Barley Half-kernels.

Distal half-kernels of barley (Hordeum vulgare L.), after imbibition for 1 day, produced a carboxypeptidase that was active on N-carbobenzoxy-l-phenylalanyl-l-alanine and on N-carbobenzoxy-l-phenylalanyl-l-phenylalanine. For the ensuing 2 days, activity increased linearly and thereafter increased at reduced rates. Electrofocusing of an imbibed half-kernel homogenate produced coincident peaks of activity on both substrates. Experiments with dissected imbibed (3 days) half-kernels showed that the enzyme arose in the aleurone layer. Enzyme production was inhibited by 6-methylpurine, cordycepin, cycloheximide, and p-fluorophenylalanine, and activity was inhibited by phenylmethylsulfonylfluoride. The enzyme did not hydrolyze endopeptidase substrates over a range of pH.Gibberellic acid accelerated the rate of release from the aleurone, but was not essential for release and did not appreciably affect the ultimate amount of carboxypeptidase produced. In these respects, the carboxypeptidase appears to be unique among the known hydrolases produced by barley aleurone tissue.

Journal Article↗

PubMed1985

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase.

Polyclonal antibodies raised against barley (1-->3,1-->4)-beta-d-glucanase, alpha-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley. In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding alpha-amylase or (1-->3,1-->4)-beta-d-glucanase, while in the aleurone alpha-amylase and (1-->3,1-->4)-beta-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1-->3,1-->4)-beta-d-glucanase, alpha-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation products were identical for each enzyme.

Journal Article↗

PubMed1986

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers.

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA(3)) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA(3). In contrast, GA(3) is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA(3) is similar to that observed for alpha-amylase. Treating aleurone layers with both GA(3) and abscisic acid prevents all the GA(3) effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA(3) response, it is concluded that the proteases represent the GA(3)-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.

Journal Article↗