Carboxypeptidase Digestion Research Articles

Demonstration of beta-tropomyosin (Tpm2) and duplication of the alpha-slow tropomyosin gene (TPM3) in Atlantic salmon Salmo salar.

Beta tropomyosin (Tpm2) is demonstrated for the first time at the protein level in a fish species, using a combination of electrophoresis, mass spectrometric peptide mapping and end-group analysis. Tpm2 accounts for 50% of the total tropomyosin in slow trunk muscle of the adult Atlantic salmon as determined by quantitative carboxypeptidase digestion and is also present in the head and pectoral fin. It is absent in the fast skeletal (lighter-toned) trunk muscle, the most abundant muscle, which is composed solely of an alpha-fast (Tpm1) isoform. In contrast to the mammalian homologues, salmon Tpm2 migrates faster than salmon Tpm1 in the presence of anionic detergent. Other distinguishing characteristics are a reduced content of cysteine (one per chain) and tyrosine (five per chain) and a unique carboxyl-terminal region (residues 276-284). Two isoforms (paralogs) of alpha-slow tropomyosin (Tpm3) having different contents of methionine and histidine exist in slow trunk muscle indicating duplication of the TPM3 gene. Minor skeletal muscles, surveyed for the first time, contain a mix of at least two tropomyosins - Tpm2 (~ 50% of total) in pectoral fin, jaw and tongue and another isoform, either Tpm1 (pectoral fin) or alpha-1-like Tpm (jaw and tongue). Cheek muscle contains Tpm1 and alpha 1-like Tpm in varying proportion depending upon the section (light or dark). Of the two tropomyosins in tongue, Tpm2 displays comparatively weaker affinity for troponin-Sepharose. A feature of the major sarcomeric tropomyosins in Atlantic salmon is a pair of neighbouring glycines situated between residues 20-90.

Graphene Oxide–Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.

The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini

Studies on Carboxypeptidase Digests of Human Hemoglobin (Antonini, E., Wyman, J., Zito, R., Rossi-Fanelli, A., and Caputo, A. (1961) J. Biol. Chem. 236, PC60–PC63) The Effect of Oxygenation on the Rate of Digestion of Human Hemoglobins by Carboxypeptidases (Zito, R., Antonini, E., and Wyman, J. (1964) J. Biol. Chem. 239, 1804–1808)

Hydrogen fluoride catalyzed migration of side chain protecting groups onto Fmoc during solid phase peptide synthesis. Characterization by CF-FAB analysis of carboxypeptidase digestions and NMR spectroscopy.

The solid-phase synthesis of the N alpha-Fmoc analog of protein kinase C substrate (PKCS, Lys-Arg-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg) was characterized by low recovery from the resin and the concomitant appearance of four impurities. FAB-MS revealed molecular weights for two of these impurities that corresponded to the desired peptide plus Tos or Bzl. The other two were justified by invoking a CO2 elimination of the Clz protecting group to yield: 1) peptide plus 2-chlorobenzyl (ClBzl) and 2) peptide plus ClBzl and Tos. A CF-FAB analysis of carboxypeptidase digestions allowed observation of peptide cleavage down to an ion corresponding to lysine, Fmoc, and the corresponding protecting group(s). These data revealed that the impurities were not the result of incomplete deprotection but the result of migration of the protecting groups to the N-terminal end of the peptide. NMR experiments were subsequently performed and revealed the exact site of substitution: the meta positions of the N-terminal Fmoc. These impurities are presumed to arise by electrophilic aromatic substitution of the fluorene group during HF treatment. The desired Fmoc analog served as a convenient, albeit low-yielding, intermediate for purification of the highly charged PKCS by preparative self-displacement HPLC.

Interaction of DDSDEEN peptide with N-CAM protein. Possible mechanism enhancing neuronal differentiation

DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide–N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.

Site-Specific Hydrogen/Deuterium Exchange Analysis of a Protein by Mass Spectrometry Coupled with Carboxypeptidase Digestion

Site-specific hydrogen/deuterium (H/D) exchange was investigated using a model protein, Escherichia coli dihydrofolate reductase, by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) coupled with two-step protease digestion by pepsin and carboxypeptidase P. Carboxypeptidase, which is generally inactive under quenching conditions (0°C and pH 2.9), produced some overlapping peptide sequences in the presence of 3.9 mM NaCl as a stabilizer. From differences in the masses of these peptides, we successfully determined the deuterium incorporation of backbone amide hydrogens of six residues—Met16 (M20 loop), Glu17 (M20 loop), Met92 (βE), Ala117 (βF-βG loop), Cys152 (βH), and Phe153 (βH)—at a higher resolution using the program Isotopica, which is capable of deconvoluting a complex isotope envelope associated with the isotope distribution of deuterated peptides. The H/D exchange kinetics data obtained were highly consistent with the local structure and fluctuation of these sites as revealed by nuclear magnetic resonance (NMR) and X-ray crystallography. These results demonstrate that MALDI MS coupled with two-step digestion is a useful tool for studying the site-specific H/D exchange of proteins without the deuterium scrambling that occurs in the gas-phase collision-induced dissociation method.

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests

A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

Small cationic protein from a marine turtle has β‐defensin‐like fold and antibacterial and antiviral activity

Egg white of marine turtle Caretta caretta contains a small cationic protein but lacks lysozyme. The protein was sequenced by a combination of sequential Edman degradation, carboxypeptidase digestion, nuclear magnetic resonance (NMR) and electrospray ionization tandem mass spectrometry. The protein contains 36 amino acid residues of which six are half-cysteines. The three-dimensional structure of the protein was deduced from two-dimensional NMR experiments and was observed to be similar to vertebrate beta-defensins. However, disulfide connectivity is C1-C6/C2-C5/C3-C4; different from that of the vertebrate beta-defensins. The protein showed strong antibacterial activity against Escherichia coli and Salmonella typhimurium. The protein also showed significant antiviral activity against an enveloped rhabdovirus, Chandipura virus, which is an emerging human pathogen. This virus is also closely related to the vesicular stomatitis virus, whose growth was also inhibited. This small cationic protein is part of the innate immunity of this organism and replaces lysozyme in the egg. It has the potential to be developed as an antibacterial and antiviral agent.

Subunit Structure of Gas Vesicles: A MALDI-TOF Mass Spectrometry Study

Many aquatic microorganisms use gas vesicles to regulate their depth in the water column. The molecular basis for the novel physical properties of these floatation organelles remains mysterious due to the inapplicability of either solution or single crystal structural methods. In the present study, some folding constraints for the ∼7-kDa GvpA building blocks of the vesicles are established via matrix-assisted laser desorption ionization time-of-flight mass spectrometry studies of intact and proteolyzed vesicles from the cyanobacterium Anabaena flos-aquae and the archaea Halobacterium salinarum. The spectra of undigested vesicles show no evidence of posttranslational modification of the GvpA. The extent of carboxypeptidase digestion shows that the alanine rich C-terminal pentapeptide of GvpA is exposed to the surface in both organisms. The bonds that are cleaved by Trypsin and GluC are exclusively in the extended N-terminus of the Anabaena flos-aquae protein and in the extended C-terminus of the Halobacterium salinarum protein. All the potentially cleavable peptide bonds in the central, highly conserved portion of the protein appear to be shielded from protease attack in spite of the fact that some of the corresponding side chains are almost certainly exposed to the aqueous medium.

C‐Terminal Sequence Analysis

Carboxy-terminal (C-terminal) sequence analysis is used for direct confirmation of the C-terminal sequence of native and expressed proteins, for detection and characterization of protein processing at the C-terminus, for identification of post-translational proteolytic cleavages, and for obtaining partial sequence information on N-terminally blocked protein samples in order to facilitate design of oligonucleotide probes for gene cloning. This unit describes an automated chemical method and a manual enzymatic (carboxypeptidase digestion) method for determining C-terminal sequence information. Carboxypeptidase digestion requires only a standard amino acid analysis method.

The multiplicity of cardiotoxins from Naja naja atra (Taiwan cobra) venom

Four novel cardiotoxins were isolated from Naja naja atra (Taiwan cobra) venom by successive separation on a SP-Sephadex C-25 column and a reverse phase column. Amino acid sequences of the cardiotoxins were determined by Edman degradation and carboxypeptidase digestion. It shows that these cardiotoxins comprise 60 amino acid residues. Comparative analyses on the amino acid sequences of cardiotoxins from the venoms of N. naja atra and other Naja species indicated that amino acid substitutions of cardiotoxin isoforms frequently occurred at positions 7–11, 27–32 and 45–47. The hypervariable segments encoded by the second and third exon of cardiotoxin genes are located at or near the tips of loop structure of cardiotoxin molecules. These results, together with the suggestions that the residues at the tips of cardiotoxins' loop structure were involved in the manifestation of the biological activities of cardiotoxins, reflect that the preferential mutations may contribute to alterations in the function of cardiotoxin molecules. Analysis on the secondary structure of pre-mRNAs of N. naja atra cardiotoxin 4 gene and N. naja sputatrix cardiotoxin 3 gene has shown that the hypervariable regions of the exon 2 pertain to form intra–exon pairings and are not involved in the formation of intron–exon pairings. Since the pairings of splice sites and gene architecture were supposed to be associated with intron–exon recognition, it is likely that the preferred loci of mutations occurring with the evolution of cardiotoxin genes would not affect the processing of cardiotoxin precursors.

Specific bonding of puromycin to full-length protein at the C-terminus.

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato : cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide

A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves. The full 5′ region of the cDNA was obtained through a RACE-PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

Hb Les Andelys [α83(F4)Leu→Pro]: A New Moderately Unstable Variant

Hb Les Andelys [α83(F4)Leu→ Proj is a mildiy unstable variant that was found during glycated hemoglobin measurement in a French family. in this hemoglobin molecule the affected site, in the α chain, and the amino acid substitution are identical to those of Hb Santa Ana, an unstable β chain variant. the structural abnormality was demonstrated by protein chemistry methods, involving, in addition to the classical techniques, a selective precipitation of the abnormal hemoglobin by isopropanol and a mass spectrometry analysis of the αT-9 peptide following carboxypeptidase digestion. DNA sequencing demonstrated that the mutation was CTG→ CCG at codon 83 of the α2 gene.

Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within alpha T9 peptide: identification of Hb Villeurbanne alpha 89 (FG1) His-->Tyr.

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the alpha-chain of human hemoglobin (residues 62-90) are difficult to identify. Despite their high m/z value (around m/z 3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated alpha T9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the alpha T9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the alpha T9 part of the sequence. Two of them, Hb Aztec [alpha 76(EF5) Met-->Thr] and Hb M-Iwate [alpha 87(F8) His-->Tyr] were already known. The third [alpha 89(FG1) His-->Tyr] was novel and named Hb Villeurbanne.

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues.

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.

Chemistry and mechanism of vanadate-promoted photooxidative cleavage of myosin.

Irradiation of the stable myosin subfragment 1(S1).MgADP.orthovanadate (Vi) complex results in oxidation of an active site serine (Ser-180) to a serine aldehyde [Cremo, C. R., Grammer, J. C., & Yount, R. G. (1989) J. Biol. Chem. 264, 6608-6611]. This photomodified S1 will reform a new MgADP.Vi complex and upon a second irradiation, the S1 heavy chain is cleaved into 21 kDa NH2-terminal and 74 kDa COOH-terminal fragments. When S1, in which the side chain of Ser-180 was tritiated, was photocleaved tritium was released from the protein suggesting that cleavage was occurring at Ser-180. The 21 kDa NH2-terminal fragment was resistant to carboxypeptidase digestion, and the 74 kDa COOH-terminal fragment yielded no sequence by Edman degradation, indicating that parts of Ser-180 went to each fragment. To identify these parts, the two cleavage fragments were isolated and chemically (21 kDa) or enzymatically (74 kDa) cleaved, and the resulting peptides were separated by reversed phase HPLC. The peptides immediately down- and up-stream from Ser-180 were isolated and the blocking groups were identified by mass spectrometry. The 21 kDa fragment peptide was blocked with a carboxamide on Glu-179 (confirmed by HPLC and capillary electrophoresis in comparison with peptide standards), while the NH2 group of Gly-181 of the 74 kDa fragment was blocked with an oxalyl group (verified by enzymatic analysis for oxalate). The side chain of Ser-180 was released as formate. O2 is required for photocleavage. Cleavage experiments in the presence of 18O2 showed one atom of 18O labeled the oxalyl group. A mechanism in which O2 adds to a free radical on the alpha-carbon of Ser-180 with a subsequent Criegee type rearrangement is proposed to explain both the kinetics and products of the photocleavage.

Amino acid sequence of ferredoxin II from the phototroph Rhodospirillum rubrum: Characteristics of a 7Fe ferredoxin

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.

A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis.

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.

Primary structures of two proteins from the venom of the Mexican red knee tarantula ( Brachypelma smithii)

Venom of the Mexican red knee tarantula ( Brachypelma smithii) was fractionated by gel filtration over Sephadex G-50 Fine. Small polypeptides present in the second and third peaks were subfractionated by cation exchange and reversed-phase FPLC. One major, basic protein was isolated and sequenced from each G-50 fraction using a gas-phase protein sequencer. Primary structures were completed and confirmed using tandem mass spectrometry and carboxypeptidase digestions. Protein 1 contains 39 residues, including six cysteine residues in three disulfide bonds. It is identical to one of the isoforms of ESTX from the venom of the tarantula Eurypelma californicum. Brachypelma smithii Protein 5 contains 34 residues, including six cysteine residues in three disulfide bonds. Disulfide bond assignments for both proteins are provided. Protein 5 shows most similarity with toxin Tx2-9 from the Brazilian ‘armed’ spider, but only displays 41% sequence identity. Similarities with other proteins are lower. Proteins 1 and 5 appear unrelated to each other.