scholarly journals LC-HRMS Screening and Identification of Novel Peptide Markers of Ricin Based on Multiple Protease Digestion Strategies

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 393 ◽  
Author(s):  
Long-Hui Liang ◽  
Chang-Cai Liu ◽  
Bo Chen ◽  
Long Yan ◽  
Hui-Lan Yu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Accurate Analysis ◽  
Ribosome Inactivating Proteins ◽  
Protease Digestion ◽  
Screening And Identification ◽  
Digestion Methods ◽  
Similar Amino Acid

Both ricin and R. communis agglutinin (RCA120), belonging to the type II ribosome-inactivating proteins (RIPs-Ⅱ), are derived from the seeds of the castor bean plant. They share very similar amino acid sequences, but ricin is much more toxic than RCA120. It is urgently necessary to distinguish ricin and RCA120 in response to public safety. Currently, mass spectrometric assays are well established for unambiguous identification of ricin by accurate analysis of differentiated amino acid residues after trypsin digestion. However, diagnostic peptides are relatively limited for unambiguous identification of trace ricin, especially in complex matrices. Here, we demonstrate a digestion strategy of multiple proteinases to produce novel peptide markers for unambiguous identification of ricin. Liquid chromatography-high resolution MS (LC-HRMS) was used to verify the resulting peptides, among which only the peptides with uniqueness and good MS response were selected as peptide markers. Seven novel peptide markers were obtained from tandem digestion of trypsin and endoproteinase Glu-C in PBS buffer. From the chymotrypsin digestion under reduction and non-reduction conditions, eight and seven novel peptides were selected respectively. Using pepsin under pH 1~2 and proteinase K digestion, six and five peptides were selected as novel peptide markers. In conclusion, the obtained novel peptides from the established digestion methods can be recommended for the unambiguous identification of ricin during the investigation of illegal use of the toxin.

scholarly journals LC-HRMS Screening and Identification of Novel Peptide Markers of Ricin Based on Multiple Protease Digestion Strategies

2019 ◽  
Author(s):  
Long-Hui Liang ◽  
Chang-Cai Liu ◽  
Bo Chen ◽  
Long Yan ◽  
Hui-Lan Yu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Accurate Analysis ◽  
Ribosome Inactivating Proteins ◽  
Protease Digestion ◽  
Screening And Identification ◽  
Digestion Methods ◽  
Similar Amino Acid

Both ricin and R. communis agglutinin (RCA120), belonging to the type II ribosome-inactivating proteins (RIPs-Ⅱ), are derived from the seeds of castor bean plant. They share very similar amino acid sequences, but ricin is much more toxic than RCA120. It is urgently necessary to distinguish ricin and RCA120 in response to public safety. Currently, mass spectrometric assays are well established for unambiguous identification of ricin by accurate analysis of differentiated amino acid residues after trypsin digestion. However, diagnostic peptides are relatively limited for unambiguous identification of trace ricin, especially in complex matrices. Here, we demonstrate a digestion strategy of multiple proteinases to produce novel peptide markers for unambiguous identification of ricin. LC-HRMS was used to verified the resulting peptides, among which only the peptides with uniqueness and good MS response were selected as peptide markers. Seven novel peptide markers were obtained from tandem digestion of trypsin and endoproteinase Glu-C in PBS buffer. From the chymotrypsin digestion under reduction and non-reduction conditions, eight and seven novel peptides were selected respectively. Using pepsin under pH 1~2 and proteinase K digestion, 6 and 5 peptides were selected as novel peptide markers. In conclusion, the obtained novel peptides from the established digestion methods can be recommended for the unambiguous identification of ricin during the investigation of illegal use of the toxin.

scholarly journals Access of proteinase K to partially translocated nascent polypeptides in intact and detergent-solubilized membranes.

1989 ◽  
Vol 108 (2) ◽  
pp. 299-307 ◽  
Author(s):  
T Connolly ◽  
P Collins ◽  
R Gilmore
Keyword(s):  
Amino Acid ◽  
Proteinase K ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Detergent Solubilization ◽  
Protease Digestion ◽  
Cytoplasmic Face ◽  
Nascent Chain ◽  
Microsomal Membranes ◽  
Vesicular Stomatitis

We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behaviour of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane.

scholarly journals Conformational Differences between Active Angiotensins and Their Inactive Precursors

Acta Naturae ◽  
2012 ◽  
Vol 4 (1) ◽  
pp. 74-77
Author(s):  
O. N. Solopova ◽  
L. P. Pozdnyakova ◽  
N. E. Varlamov ◽  
M. N. Bokov ◽  
E. V. Morozkina ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Polypeptide Chain ◽  
Peptide Conformation ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
High Affinity ◽  
Peptide Core ◽  
Similar Amino Acid

The peptide conformation in the context of a protein polypeptide chain is influenced by proximal amino acid residues. However, the mechanisms of this interference remain poorly understood. We studied the conformation of angiotensins 1, 2 and 3, which are produced naturally in a sequential fashion from a precursor protein angiotensinogen and contain an identical peptide core structure. Using the example of angiotensins 1, 2 and 3, it was shown that similar amino acid sequences may have significant conformational differences in various molecules. In order to assess the conformational changes, we developed a panel of high-affinity mouse monoclonal antibodies against angiotensins 1, 2 and 3 and studied their cross-reactivity in indirect and competitive ELISAs. It was found that the conformations of inactive angiotensin1 and the corresponding fragment of angiotensinogen are similar; the same is true for the conformations of active angiotensins 2 and 3, whereas the conformations of homologous fragments in the active and inactive angiotensins differ significantly.

scholarly journals Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

2005 ◽  
Vol 71 (7) ◽  
pp. 3420-3426 ◽  
Author(s):  
Helena Gradišar ◽  
Jožica Friedrich ◽  
Igor Križaj ◽  
Roman Jerala
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Paecilomyces Marquandii ◽  
Molecular Masses ◽  
Terminal Amino ◽  
Hydrolysis Of

ABSTRACT Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.

Computational Analysis of Therapeutic Enzyme Uricase from Different Source Organisms

2020 ◽  
Vol 17 (1) ◽  
pp. 59-77
Author(s):  
Anand Kumar Nelapati ◽  
JagadeeshBabu PonnanEttiyappan
Keyword(s):  
Uric Acid ◽  
Amino Acid ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
Protein Sequences ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Multiple Sequence ◽  
Physiochemical Properties ◽  
Pharmaceutical Industries

Background:Hyperuricemia and gout are the conditions, which is a response of accumulation of uric acid in the blood and urine. Uric acid is the product of purine metabolic pathway in humans. Uricase is a therapeutic enzyme that can enzymatically reduces the concentration of uric acid in serum and urine into more a soluble allantoin. Uricases are widely available in several sources like bacteria, fungi, yeast, plants and animals.Objective:The present study is aimed at elucidating the structure and physiochemical properties of uricase by insilico analysis.Methods:A total number of sixty amino acid sequences of uricase belongs to different sources were obtained from NCBI and different analysis like Multiple Sequence Alignment (MSA), homology search, phylogenetic relation, motif search, domain architecture and physiochemical properties including pI, EC, Ai, Ii, and were performed.Results:Multiple sequence alignment of all the selected protein sequences has exhibited distinct difference between bacterial, fungal, plant and animal sources based on the position-specific existence of conserved amino acid residues. The maximum homology of all the selected protein sequences is between 51-388. In singular category, homology is between 16-337 for bacterial uricase, 14-339 for fungal uricase, 12-317 for plants uricase, and 37-361 for animals uricase. The phylogenetic tree constructed based on the amino acid sequences disclosed clusters indicating that uricase is from different source. The physiochemical features revealed that the uricase amino acid residues are in between 300- 338 with a molecular weight as 33-39kDa and theoretical pI ranging from 4.95-8.88. The amino acid composition results showed that valine amino acid has a high average frequency of 8.79 percentage compared to different amino acids in all analyzed species.Conclusion:In the area of bioinformatics field, this work might be informative and a stepping-stone to other researchers to get an idea about the physicochemical features, evolutionary history and structural motifs of uricase that can be widely used in biotechnological and pharmaceutical industries. Therefore, the proposed in silico analysis can be considered for protein engineering work, as well as for gout therapy.

scholarly journals Bile-pigment formation from different leghaemoglobins. Methine-bridge specificity of coupled oxidation

1978 ◽  
Vol 176 (2) ◽  
pp. 359-364 ◽  
Author(s):  
Päivi Lehtovaara ◽  
Ulla Perttilä
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Broad Bean ◽  
Amino Acid Sequences ◽  
Second Step ◽  
Bile Pigment ◽  
Site Specificity ◽  
Amino Acid Residues ◽  
Reaction Step ◽  
Pigment Formation

The coupled oxidation of leghaemoglobins with O2 and ascorbate yielded oxyleghaemoglobin in the first reaction step, and the second step was the degradation of haem characterized by an A675 increase. Leghaemoglobins were degraded to biliverdin isomers specifically, depending on the structure of the protein. The main leghaemoglobin components of Glycine (soya bean) and Phaseolus (kidney bean) were degraded to biliverdin mixtures containing about 50% of the β-form, about 30% of the α-form and about 20% of the δ-isomer, whereas the leghaemoglobin I components of Vicia (broad bean) and Pisum (pea) were degraded almost exclusively to the β-isomer, with traces of the α-isomer. The amino acid sequences of Glycine and Phaseolus leghaemoglobins resemble each other, as do those of Vicia and Pisum. The site specificity of bile-pigment formation from leghaemoglobins can be tentatively explained by specific differences in the amino acid sequences at those regions of the polypeptide chain that are in the vicinity of the appropriate methine bridges. The ligand-binding site in different leghaemoglobins may be outlined on the basis of the present results, supposing that the haem is degraded when a reduction product of haem-bound O2 reacts with a methine bridge of the haem, and that the bridge specificity is regulated by hindering amino acid residues that determine the location of the bound O2. The residue phenylalanine-CD1 appears to be further away from the haem plane or in a markedly more flexible position in leghaemoglobins than in mammalian globins. The haem-bound oxygen atom B, in Fe–O(A)–O(B), seems to be free to rotate in all directions except that of the γ-bridge in Glycine and Phaseolus leghaemoglobins, but its position in Vicia and Pisum leghaemoglobin I might be restricted to the direction of the β-methine bridge.

scholarly journals Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

1994 ◽  
Vol 299 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Y Deyashiki ◽  
A Ogasawara ◽  
T Nakayama ◽  
M Nakanishi ◽  
Y Miyabe ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Human Bile ◽  
Coding Regions ◽  
Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

scholarly journals The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

1973 ◽  
Vol 133 (4) ◽  
pp. 805-819 ◽  
Author(s):  
Francesco Bossa ◽  
Donatella Barra ◽  
Massimo Carloni ◽  
Paolo Fasella ◽  
Francesca Riva ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Primary Structure ◽  
Aspartate Aminotransferase ◽  
Heart Muscle ◽  
Science And Technology ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Lending Library

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

scholarly journals Diarrhea-Inducing Activity of Avian Rotavirus NSP4 Glycoproteins, Which Differ Greatly from Mammalian Rotavirus NSP4 Glycoproteins in Deduced Amino Acid Sequence, in Suckling Mice

2002 ◽  
Vol 76 (11) ◽  
pp. 5829-5834 ◽  
Author(s):  
Yoshio Mori ◽  
Mohammed Ali Borgan ◽  
Naoto Ito ◽  
Makoto Sugiyama ◽  
Nobuyuki Minamoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Suckling Mice

ABSTRACT Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences. The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4.

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