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PubMed1980

The mechanism of action of penicillin. Penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase.

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of 14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore, the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent for peptides.

Amino Acid Sequence↗

PubMed1995

[The interconnection between the activity of carboxypeptidase N and angiotensin-converting enzyme].

Inhibition of the angiotensin-converting enzyme by guanidino-ethylmercaptosuccinic acid, a highly specific inhibitor of carboxypeptidase H, and inhibition of carboxypeptidase H by captopril, a highly specific inhibitor of the angiotensin-converting enzyme, in vivo were studied. Guanidinoethylmercaptosuccinic acid and captopril had no effect either on the angiotensin-converting enzyme or the carboxypeptidase H activities in vitro. The putative mechanisms of the inhibitors effects of the enzyme activity are discussed.

Angiotensin-Converting Enzyme Inhibitors↗

PubMedDate not supplied

[The effect of ethanol on the activity of soluble and membrane-bound carboxypeptidase H in areas of the rat brain during immobilization stress].

The activity of carboxypeptidase H was inhibited dissimilarly in some brain regions of rats after combined effects of ethanol and immobilization stress. This dissimilar effect of ethanol and stress on activity of soluble and membrane-bound carboxypeptidases was detected in the midbrain, hippocamp, and striatum. The role of carboxypeptidase H in the stress-protective effect of ethanol is discussed.

Animals↗

PubMedDate not supplied

[Effect of ethanol on carboxypeptidase H in the rat brain].

It is established that under a single-dose introduction of ethanol activity of carboxypeptidase H increases. During chronic consumption of ethanol changes of enzyme activity was less than during a single-dose introduction of ethanol. Most considerable changes in carboxypeptidase H activity are observed in the pituitary gland, hypothalamus and striatum. Ethanol effect on the activity of membrane-bound and soluble forms of enzyme was distinguished. It is supposed that carboxypeptidase H is involved into the formation of response to ethanol.

Animals↗

PubMed1976

Carboxypeptidase B-like and trypsin-like activities in isolated rat pancreatic islets.

Trypsin-like and carboxypeptidase B-like proteinases are believed to play important roles in the conversion of proinsulin into insulin as well as in the intracellular processing of a variety of other precursor forms. To facilitate the study of these enzymes we have developed sensitive methods for their detection in tissue preparations and incubation media. Studies with rat islet homogenates indicate the presence of both trypsin-like and carboxypeptidase B-like activities with slightly acidic pH optima. The trypsin-like activity was activated by thiols and inhibited by several thiol reagents but the carboxypeptidase was inhibited only by chelating agents. These properties suggest that these enzymes are related to the tissue cathepsins. Additional experimental approaches to the problems of positively identifying and localizing converting enzymes at the subcellular level are briefly discussed.

Animals↗

PubMed2006

Drosophila S2 cells produce multiple forms of carboxypeptidase D with different intracellular distributions.

Carboxypeptidase D (CPD) functions in the processing of proteins that transit the secretory pathway, and is present in all vertebrates examined as well as Drosophila. Several forms of CPD mRNA were previously found in Drosophila that resulted from differential splicing of the gene. In the present study, Northern blot, reverse transcriptase PCR, and Western blot analysis showed that each splice variant occurs in a single cell type, the Drosophila-derived Schneider 2 (S2) cell line. The short forms containing a single carboxypeptidase domain were secreted from the S2 cells while the long forms containing three carboxypeptidase domains, a transmembrane domain, and one of two different cytosolic tails were retained in the cell. To investigate the role of the two different C-terminal tail sequences (tail-1 and tail-2) that result from the differential splicing within exon 8, constructs containing a reporter protein (albumin) attached to the transmembrane domain and tail-1 or tail-2 of CPD were expressed in S2 cells and a mouse pituitary cell line (AtT20 cells). Immunofluorescence analysis revealed different intracellular distributions of the two constructs, with the tail-2 construct showing considerable overlap with a Golgi marker. The two C-terminal tail sequences also resulted in different internalization efficiencies from the cell surface in both cell lines. Interestingly, the distribution and routing of the tail-2 form of Drosophila CPD in the AtT20 cells are similar to the previously characterized endogenous mouse CPD protein, indicating that the elements for this trafficking have been conserved between Drosophila and mammals.

Alternative Splicing↗

PubMed1984

Inhibition of brain tubulinyl-tyrosine carboxypeptidase by endogenous proteins.

When a 25-50% ammonium-sulphate-insoluble fraction from a bovine brain preparation was chromatographed on a cellulose phosphate column, several protein fractions which inhibit the activity of tubulinyl-tyrosine carboxypeptidase were obtained. One of these fractions exhibited activity of fructose-bisphosphate aldolase (EC 4.1.2.13) and the enzyme accounted for more than 95% of the protein of this fraction as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The inhibitory activities of the two protein fractions which had the highest activity per mg of protein were practically abolished by pretreatment with pronase; preincubation with trypsin, on the other hand, caused only a partial inactivation of the inhibitors. The inhibitory activities were little affected by heating at 90 degrees C for 5 min. Preincubation with purified tubulinyl-tyrosine carboxypeptidase caused a great decrease of the inhibitory activities of these two fractions, leaving open the possibility that these inhibitors act as substrates of the carboxypeptidase.

Animals↗

PubMed1996

Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae.

A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.

Animals↗

PubMed2000

Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane.

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed.

Animals↗

PubMed1978

Release of [14C]tyrosine from tubulinyl-[14C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase.

The carboxypeptidase previously described that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mM MgCl2 or 30 mM KCl. Mercaptoethanol (50 mM), colchicine (0.2 mM) and tyrosine (0.2 mM) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mM), deoxycholate (0.5%), CuCl2 (0.1 mM), ZnCl2 (0.1 mM) and NaCl or KCl (240 mM) had a strong inhibitory effect. The optimal pH of this enzyme was 6.3--7. A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg2+, K+ and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl2 was 3--20 mM and of KCl was 10--30 mM. For ADP the maximal act;vity was at 0.3 mM or higher. An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not.

Animals↗

PubMed1989

Inactivation of carboxypeptidase Y by mutational removal of the putative essential histidyl residue.

Carboxypeptidase Y is a serine carboxypeptidase assumed to contain a catalytic triad similar to the serine endopeptidases. On the basis of the homology between various serine carboxypeptidases His-397 is suspected to be part of the catalytic triad. To test this it was exchanged with Ala and Arg by site-directed mutagenesis of the cloned PRC1 gene. The catalytic efficiency of the mutant enzymes were reduced by a factor of 2 X 10(4) and 7 X 10(2), respectively, confirming the key role of His-397 in catalysis. Treatment of Ala-397-CPD-Y with Hg++ or CNBr, hence modifying Cys-341 located in the vicinity of the active site abolished the residual activity of the enzyme, indicating an additional involvement of this residue in catalysis.

Amino Acid Sequence↗

PubMed1994

Immobilized carboxypeptidase N. A potent bioreactor and specific adsorbent for peptides.

Carboxypeptidase N-Sepharose was prepared by covalent immobilization of purified human plasma carboxypeptidase N. More than 98% of the carboxypeptidase N was immobilized; 42% of the applied activity can be detected on the support. The column has excellent capabilities to quantitatively remove carboxy-terminal basic amino acids from peptides, as is demonstrated using the synthetic peptide substrate hippuryl-L-arginine and the nonapeptide bradykinin, and remains stable for several months. In contrast with apocarboxypeptidase B-Sepharose, apocarboxypeptidase N-Sepharose poorly binds its substrates.

Adsorption↗

PubMed2002

A germination-related gene encoding a serine carboxypeptidase is expressed during the differentiation of the vascular tissue in wheat grains and seedlings.

Carboxypeptidases expressed in the aleurone layer participate in the mobilization of endosperm storage proteins during cereal grain germination. The genes encoding these proteins are also expressed in the scutellum of germinating grains, but their function in this organ is not yet clear. We have analyzed the expression of a carboxypeptidase III (CPIII) gene in germinating wheat (Triticum aestivum L.) grains. CPIII transcripts accumulated transiently in the scutellum showing a maximum at 2-3 days after imbibition and were exclusively localized to the scutellar vascular tissue. The analysis of CPIII expression in developing shoots and roots from growing seedlings confirmed the localization of CPIII transcripts to differentiating vascular tissue. The TUNEL assay detected in situ nuclear DNA fragmentation in cells showing CPIII expression, indicating that they undergo programmed cell death. Relative RT-PCR analysis showed that the CPIII gene expressed at high level in aleurone cells is the one expressed in vegetative tissues, and allowed the use of this gene as a molecular marker of tracheary element differentiation in wheat seedlings. These results are indicative of the involvement of serine carboxypeptidases in programmed cell death during the development of the vascular tissue in wheat, a new role for these enzymes, besides the mobilization of starchy-endosperm proteins during germination.

Carboxypeptidases↗

PubMed2001

Characterization and localization of a wound-inducible type I serine-carboxypeptidase from leaves of tomato plants (Lycopersicon esculentum Mill.).

During the course of characterization of the wound-response related proteins from tomato (Lycopersicon esculentum Mill.) leaves, a serine carboxypeptidase (EC 3.4.16.1) was identified. An increase in peptidase activity in response to wounding, and the isolation of a protein with carboxypeptidase (CP) activity from tomato leaves had been reported previously, but the mRNA coding for the enzyme was not identified. We now report the isolation of a tomato leaf type I serine-CP cDNA whose corresponding mRNA is induced by wounding, systemin and methyl jasmonate. The protein sequence deduced from the cDNA exhibits homology to tomato CP, and barley and rice type I CPs. Southern blot results indicated that the CP gene is probably a member of a small gene family. Tomato CP mRNA was detected within 3 h after wounding, or treatment with systemin or methyl jasmonate. Employing Western blot analysis, CP protein was shown to increase 12 h after the treatments. Using the tomato def1 mutant, we have demonstrated that a functional octadecanoid pathway is necessary for CP transcription in response to wounding. Carboxypeptidase protein was immunolocalized as protein aggregates within the central vacuoles of palisade mesophyll cells as well as in vascular parenchyma where it had previously been found. Double labeling using antibodies specific for CP and inhibitor II indicated that the two proteins are colocalized in the vacuolar aggregates. Tomato CP is a member of the "late wound-inducible genes" whose mRNAs increase 4-12 h following wounding, in contrast to several "early wound-inducible genes", whose mRNAs appear within 30 min. The data support a role for the enzyme in protein turnover that occurs systemically in leaf cells in response to wounding.

Amino Acid Sequence↗

PubMed2005

The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica).

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the "alpha/beta hydrolase fold". The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.

Amino Acid Sequence↗

PubMed2004

Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus.

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 degrees C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 degrees C for 1 h, and up to 50 degrees C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl- l-tyrosyl- l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K(m), V(max), K(cat), and K(cat) /K(m) values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 degrees C were 0.86 mM, 0.917 mM min(-1), 291 s(-1), and 339 mM(-1 )s(-1), respectively.

Carboxypeptidases↗

PubMed1985

Use of carboxypeptidase Y in studies of the mode of insertion of cytochrome b5 into lipid vesicles.

Carboxypeptidase Y preparations from baker's yeast have been found to exhibit endopeptidase activity when cytochrome b5 was used as substrate. As the susceptibility of cytochrome b5 to attack by carboxypeptidase Y has been used to distinguish between two modes of insertion of cytochrome b5 into lipid bilayer, one which has the C terminal buried in the lipid bilayer and one which has a free C terminal, caution should be taken when employing carboxypeptidase Y preparations for this type of studies.

Animals↗

PubMed1989

The integrity of tubulin molecule is not required for the activity of tubulin carboxypeptidase.

Tubulin dimer, alpha-tubulin subunit, and C-terminal peptides obtained from the alpha-tubulin subunit were compared in their capabilities to act as substrates of tubulin carboxypeptidase. The results obtained indicate that the enzyme does not require the beta-tubulin subunit to release tyrosine from alpha-tubulin. The 17-Kd C-terminal peptide of the alpha-tubulin subunit was obtained and it was detyrosinated at the same rate as tubulin dimer. A smaller C-terminal peptide of 2.8-3.7 Kd showed a lower capability to act as substrate. Similar results were obtained with pancreatic carboxypeptidase A. From the analysis of the results we consider that an optimal activity of the tubulin carboxypeptidase depends mainly on the accessibility of the C-terminal end of alpha-tubulin.

Amino Acid Sequence↗