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 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 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.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

N-Glycans Protect Proteins from Protease Digestion through Their Binding Affinities for Aromatic Amino Acid Residues

2000 ◽  
Vol 127 (3) ◽  
pp. 427-433 ◽  
Author(s):  
T. Nishiyama ◽  
N. Kimura ◽  
Y. Jitsuhara ◽  
M. Uchida ◽  
F. Ochi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
Protease Digestion

scholarly journals The N-terminal amino acid sequence of pig kidney endopeptidase-24.11 shows homology with pro-sucrase-isomaltase

1986 ◽  
Vol 240 (1) ◽  
pp. 305-308 ◽  
Author(s):  
I S Fulcher ◽  
D J Pappin ◽  
A J Kenny
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Consensus Sequence ◽  
Terminal Segment ◽  
Amino Acid Residues ◽  
Endopeptidase 24.11 ◽  
Cytoplasmic Face ◽  
Arginine Residues ◽  
Terminal Amino ◽  
Hydrophobic Anchor

Endopeptidase-24.11 (EC 3.4.24.11), a widely distributed ectoenzyme, was isolated from pig kidneys by detergent solubilization of membranes and immuno-affinity chromatography. In all, 12 preparations of the enzyme were submitted to solid-phase sequencing, yielding a consensus sequence of 25 amino acid residues of the N-terminal segment. Some samples were treated with either trypsin or Staphylococcus aureus V8 proteinase before sequencing. There were four lysine and one arginine residues in the first nine positions. This segment was susceptible to hydrolysis by trypsin and, in some samples, to endogenous proteinases. From residue 19 onwards, the sequence became intensely hydrophobic. There was a striking homology with the N-terminal sequence of pro-sucrase-isomaltase. From Lys7 to Leu20 there were seven identical amino acid residues and four conservative substitutions. We suggest that endopeptidase-24.11 is topologically similar to this glycosidase, the N-terminus at the cytoplasmic face and hydrophobic segment serving the roles of both signal peptide and hydrophobic anchor.

scholarly journals Yeast Sup35 Prion Structure: Two Types, Four Parts, Many Variants

2019 ◽  
Vol 20 (11) ◽  
pp. 2633 ◽  
Author(s):  
Alexander Dergalev ◽  
Alexander Alexandrov ◽  
Roman Ivannikov ◽  
Michael Ter-Avanesyan ◽  
Vitaly Kushnirov
Keyword(s):  
Amino Acid ◽  
Ex Vivo ◽  
Mass Spectrometric ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Structural Variants ◽  
Spectrometric Identification ◽  
Mass Spectrometric Identification ◽  
Nonsense Suppressor ◽  
Different Origin

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants the Sup35 amino acid residues 2–32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73–124, 125–153, and 154–221, but their presence differed between [PSI+] isolates. Two distinct digestion patterns were observed for region 2–72, which always correlated with the “strong” and “weak” [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.

scholarly journals Studies of the limited degradation of mucus glycoproteins. The effect of dilute hydrogen peroxide

1983 ◽  
Vol 211 (2) ◽  
pp. 323-332 ◽  
Author(s):  
J M Creeth ◽  
B Cooper ◽  
A S R Donald ◽  
J R Clamp
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Simple Structure ◽  
Peptide Bond ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Oligosaccharide Moiety ◽  
Small Gain ◽  
Mucus Glycoproteins

1. The action of dilute H2O2 on a series of ovarian-cyst glycoproteins and glycopolypeptides was investigated. 2. Both native glycoproteins and the glycopolypeptides were carbohydrate-rich, of relatively low molecular weight and of simple structure. 3. At pH 5.6 and 37 degrees C, exposure to H2O2 for a limited time brought about a partial degradation, the molecular weight being decreased by 2-4-fold. 4. Carbohydrate analysis showed very little change in the oligosaccharide moiety, apart from a small decrease in sialic acid in some samples. 5. Amino acid analysis showed minor changes in serine, threonine and proline contents, but almost total loss of histidine. Concomitantly, there was a small gain in aspartic acid. 6. Myosin, examined at both pH 5.7 and 6.7, exhibited generally similar behaviour, there being losses of other amino acid residues as well as histidine: the viscosity was decreased to a low value, and a range of peptides of widely varying size was produced. 7. It is suggested that attack on the histidine residue, with partial conversion into aspartic acid, is accompanied by scission of the histidyl peptide bond.

The amino acid sequence of wheat β-purothionin

1976 ◽  
Vol 54 (10) ◽  
pp. 835-842 ◽  
Author(s):  
A. S. Mak ◽  
B. L. Jones
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Basic Protein ◽  
Viscum Album ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Basic Proteins ◽  
Considerable Homology

The complete amino acid sequence of β-purothionin, a low molecular weight, very basic, protein isolated from wheat endosperm material, has been determined. β-purothionin is toxic to some bacteria, to yeasts, and to animals when injected. The protein contains 45 amino acid residues and has a molecular weight of 4913. The 8 cysteine and 10 basic residues are distributed throughout the molecule. The primary structure of the protein shows considerable homology to those of the viscotoxins, which are toxic, small, basic proteins found in the leaves and stems of European mistletoe (Viscum album L.).

scholarly journals The Effect of a Peptide Substrate Containing an Unnatural Branched Amino Acid on Chymotrypsin Activity

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 242
Author(s):  
Yuuki Yamawaki ◽  
Tomoki Yufu ◽  
Tamaki Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Unnatural Amino Acids ◽  
Side Chain ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Reaction Velocity ◽  
Natural Amino Acids

7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific proteases, such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the protease toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but chymotrypsin showed the highest specificity for this compound. These results demonstrated that the substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but also by the structural expanse of amino acid residues in the substrate.

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