scholarly journals Isolation and characterization of a polymerized prion protein

2002 ◽  
Vol 364 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Bao-Yuan LU ◽  
Jui-Yoa CHANG
Keyword(s):  
Prion Protein ◽  
Cd Spectroscopy ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Proteinase K ◽  
Guanidinium Chloride ◽  
Isolation And Characterization ◽  
Size Exclusion Hplc ◽  
Β Sheet

A polymerized form of recombinant mouse prion protein (mPrP) domain 23–231 [mPrP-(23–231)], designated mPrP-z, was generated at acidic pH (pH 2–5) in the presence of selected concentrations of denaturant (2M guanidinium chloride or 5M urea). This isoform of mPrP is stable in acidic solution after removal of denaturant. It can be isolated and purified using reversed-phase HPLC or size-exclusion HPLC. mPrP-z bears structural properties that partially resemble those of scrapie prion. Unlike the native mPrP-(23–231) (mPrP-N), mPrP-z exhibits a high content of β-sheet structure, as shown by CD spectroscopy, and exists as an oligomer with an approximate molecular mass of 340000Da, as measured by light scattering. However, similarly to mPrP-N, mPrP-z contains the intact disulphide bond and is sensitive to digestion by proteinase K.

scholarly journals Copper induces increased beta-sheet content in the scrapie-susceptible ovine prion protein PrPVRQ compared with the resistant allelic variant PrPARR

2004 ◽  
Vol 380 (1) ◽  
pp. 273-282 ◽  
Author(s):  
Edmond WONG ◽  
Alana M. THACKRAY ◽  
Raymond BUJDOSO
Keyword(s):  
Amino Acid ◽  
Prion Protein ◽  
Metal Ion ◽  
Cd Spectroscopy ◽  
Proteinase K ◽  
Copper Binding ◽  
Protein Amino Acid ◽  
Allelic Variants ◽  
Β Sheet

Prion diseases are characterized by conformational change in the copper-binding protein PrP (prion protein). Polymorphisms in ovine PrP at amino acid residues 136, 154 and 171 are associated with variation in susceptibility to scrapie. PrPVRQ [PrP(Val136/Arg154/Gln171)] or PrPARQ [PrP(Ala136/Arg154/Gln171)] animals show susceptibility to scrapie, whereas those that express Ala136/Arg154/Arg171 (PrPARR) show resistance. Results are presented here that show PrPVRQ and PrPARR display different conformational responses to metal-ion interaction. At 37 °C copper induced different levels of β-sheet content in the allelic variants of ovine full-length prion protein (amino acid 25–232). PrPVRQ showed a significant increase in β-sheet content when exposed to copper at 37 °C, whereas PrPARR remained relatively unchanged. The conversion of α-helical PrPVRQ to β-sheet form was shown by CD spectroscopy and the decreased binding of C-terminal specific monoclonal anti-PrP antibodies. This conversion to an increased β-sheet form did not occur with truncated PrPVRQ (amino acids 89–233), which demonstrates that additional metal-binding sites outside of the N-terminus may not overtly influence the overall structure of ovine PrP. Despite the difference in β-sheet content, both the scrapie-susceptible and -resistant allelic forms of ovine PrP acquired resistance to proteinase K digestion following exposure to copper at 37 °C, suggesting the potential for disease-associated PrPARR to accumulate in vivo. Our present study demonstrates that allelic variants of ovine PrP differ in their structure and response to the interaction with copper. These observations will contribute to a better understanding of the mechanism of susceptibility and resistance to prion disease.

Purification and partial amino acid sequence of thuricin S, a new anti-Listeriabacteriocin from Bacillus thuringiensis

2007 ◽  
Vol 53 (2) ◽  
pp. 284-290 ◽  
Author(s):  
Sonia Chehimi ◽  
François Delalande ◽  
Sophie Sablé ◽  
Mohamed-Rabeh Hajlaoui ◽  
Alain Van Dorsselaer ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Proteinase K ◽  
Terminal Sequence ◽  
Partial Amino Acid Sequence ◽  
Ph Values ◽  
Heat Stable ◽  
Isolation And Characterization ◽  
Terminal Amino ◽  
Edman Sequencing

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3–10.5). The monoisotopic mass of thuricin S purified by high perfomance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.

scholarly journals The Prion Protein Octarepeat Domain Forms Transient β-sheet Structures Upon Residue-Specific Cu(II) and Zn(II) Binding

2021 ◽  
Author(s):  
Maciej Gielnik ◽  
Aneta Szymanska ◽  
Xiaolin Dong ◽  
Jyri Jarvet ◽  
Zeljko M. Svedruzic ◽  
...  
Keyword(s):  
Metal Ions ◽  
Prion Protein ◽  
Metal Binding ◽  
Cd Spectroscopy ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Amyloid Formation ◽  
Spectroscopy Data ◽  
Spongiform Encephalopathies ◽  
Β Sheet

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in the protein misfolding, and metal imbalance may be part of TSE pathologies. PrPC is a combined Cu(II) and Zn(II) metal binding protein, where the main metal binding site is located in the octarepeat (OR) region. Here, we used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Upon metal binding, the OR region seems to adopt a transient antiparallel β-sheet hairpin structure. Fluorescence spectroscopy data indicates that under neutral conditions, the OR region can bind both Cu(II) and Zn(II) ions, whereas under acidic conditions it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of both metal ions to the OR region results in formation of β-hairpin structures. As formation of β-sheet structures is a first step towards amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSEs.

scholarly journals Purification and characterization of cystatin from duck egg white.

1995 ◽  
Vol 42 (3) ◽  
pp. 351-356 ◽  
Author(s):  
M Warwas ◽  
J Gburek ◽  
J Osada ◽  
K Gołab
Keyword(s):  
Egg White ◽  
Amino Acid Sequences ◽  
Size Exclusion ◽  
Molecular Forms ◽  
Sds Page ◽  
Peptidase Inhibitor ◽  
Size Exclusion Hplc ◽  
Duck Egg ◽  
Chicken Cystatin

It is the second peptidase inhibitor, after ovostatin, which showing the same antipapain activity in egg white in different avian species implies differences in amino-acid sequences. Cystatin from duck egg white was purified by carboxymethylpapain affinity chromatography and size-exclusion HPLC. The purified inhibitor which showed partial identity in the immunodiffusion test with chicken egg white cystatin, had an apparent molecular mass of 9.3 kDa as determined by SDS/PAGE. IEF analysis revealed five molecular forms of pI in the range 7.8-8.4. The obtained cystatin was neither glycosylated nor phosphorylated as it is in the case of chicken cystatin. The determined Ki (0.005 +/- 0.001 nM) was similar to that reported for human and chicken cystatin C.

scholarly journals Isolation, Identification and Characterization of Degradation Impurity of Atorvastatin in Fixed Dose Combination of Atorvastatin and Ezetimibe

2019 ◽  
Vol 11 (05) ◽  
Author(s):  
Rajesh Desai ◽  
Suresh Koradia
Keyword(s):  
Reversed Phase ◽  
Fixed Dose Combination ◽  
Fixed Dose ◽  
Forced Degradation ◽  
Preparative Hplc ◽  
Atorvastatin Calcium ◽  
Isolation And Characterization ◽  
Dose Combination ◽  
Liquid Chromatographic

The objective of this study is to isolation and characterization of unknown degradation product of Atorvastatin calcium in combination formulation product with Ezetimibe by using modern techniques of separation and aracterization. An unknown impurity is generating during a forced degradation study of Atorvastatin and Ezetimibe fixed-dose combination tablets. By using the gradient reversed-phase high-pressure liquid chromatographic method, unknown degradation impurity was detected and quantified in the range of 0.05% to 0.2% of Atorvastatin. The impurity was enriched by extreme oxidation degradation of Atorvastatin and isolated through preparative HPLC. The structure of the impurity was characterized by mass and NMR spectrum.

scholarly journals Isolation and characterization of a digoxin-like immunoreactive substance from human urine by affinity chromatography

1997 ◽  
Vol 43 (8) ◽  
pp. 1416-1420 ◽  
Author(s):  
Claudio De Angelis ◽  
Massimiliano Riscazzi ◽  
Riccardo Salvini ◽  
Alfonso Piccoli ◽  
Claudio Ferri ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Human Urine ◽  
Biological Fluids ◽  
Active Material ◽  
Reversed Phase ◽  
Cross Reactivity ◽  
Linear Gradient ◽  
Isolation And Characterization ◽  
Dog Kidney

Abstract A series of observations has suggested that one or more digoxin-like immunoreactive substances (DLIS) in biological fluids is able to cross-react with the antidigoxin antibody. Whether this substance is the endogenous inhibitor of Na+/K+ ATPase has not been well established. The aim of this study was to identify and characterize DLIS from human urine. Treated urine from healthy men was run on an affinity chromatography column at a flow rate of 1 mL/min in which the ligand was an antibody (antiserum) to digoxin. Eluates from affinity chromatography were applied onto analytical reversed-phase HPLC. The active material was eluted with a linear gradient of acetonitrile (from 350 to 650 mL/L) and water. A second step in HPLC was carried out isocratically with 280 mL/L acetonitrile in water. We found a single peak showing cross-reactivity with antidigoxin antibody as measured by RIA. It showed the same retention time as that of a digoxin calibrator. This highly purified substance is able to displace [3H]ouabain from dog kidney-derived Na+/K+ ATPase, to inhibit Na+/K+ ATPase activity as measured by the86Rb+ uptake in red blood cells and by coupled enzyme assay. Our results are consistent with the hypothesis that DLIS, as isolated by this particular digoxin antibody, is a single substance and an inhibitor of Na+/K+ ATPase.

scholarly journals False Biomarker Discovery due to Reactivity of a Commercial ELISA for CUZD1 with Cancer Antigen CA125

2014 ◽  
Vol 60 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Ioannis Prassas ◽  
Davor Brinc ◽  
Sofia Farkona ◽  
Felix Leung ◽  
Apostolos Dimitromanolakis ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Size Exclusion ◽  
Ductal Adenocarcinoma ◽  
Elisa Assay ◽  
Expression Systems ◽  
Cancer Antigen 125 ◽  
Cancer Antigen ◽  
Specific Proteins ◽  
Size Exclusion Hplc

Abstract BACKGROUND By using proteomics and bioinformatics, we have previously identified a group of highly pancreas-specific proteins as candidate pancreatic ductal adenocarcinoma (PDAC) biomarkers. With the use of commercially available ELISAs, the performance of some of these candidates was initially evaluated in a relatively small serum cohort (n = 100 samples). This phase revealed that CUB and zona pellucida-like domains protein 1 (CUZD1) may represent a new, promising PDAC biomarker. METHODS We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay. CUZD1 was expressed in house in both bacteria and yeast expression systems. Recombinant CUZD1 and biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards, were analyzed by Western blot, size exclusion HPLC, and mass spectrometry (LC-MS Orbitrap). RESULTS We confirmed that instead of CUZD1, the commercial assay is recognizing a nonhomologous, known cancer antigen [cancer antigen 125 (CA125)]. CONCLUSIONS We conclude that poor characterization of commercial ELISA assays is a factor that could lead to false biomarker discovery. To our knowledge, this is the first report documenting that a commercial ELISA marketed for one analyte (CUZD1) may, in fact, recognize a different, nonhomologous antigen (CA125).

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