Effect of Lyotropic Salts on the Stability of a Subtilisin-Like Proteinase from a Psychrotrophic Vibrio-Species, Proteinase K and Aqualysin I.

We compared the morphology and stability of hybridization signals between paraffin sections of rat retina fixed with commonly used 4% paraformaldehyde/PBS and those fixed with a fixative containing glutaraldehyde in in situ hybridization histochemistry, using a digoxigenin-labeled RNA probe complementary for beta-galactoside alpha 2,6-sialyltransferase mRNA. Retinal detachment was frequently observed in the sections fixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfactorily preserved in those fixed with either 0.5% glutaraldehyde, 4% paraformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde, it was difficult to determine the most appropriate length of proteinase K digestion of tissue sections for facilitating probe penetration, since the optimal time for definite hybridization was variable among the retinal cells in heterogeneous layers. By addition of glutaraldehyde to paraformaldehyde or with glutaraldehyde alone, it was easy to establish the appropriate time for the unmasking procedure, since intense mRNA signals were constant throughout the retina by proteinase K digestion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not only the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxigenin-labeled RNA probes.

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Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species . Comparison with proteinase K and aqualysin I

European Journal of Biochemistry ◽

10.1046/j.1432-1327.1999.00205.x ◽

1999 ◽

Vol 260 (3) ◽

pp. 752-760 ◽

Cited By ~ 45

Author(s):

Magnus M. Kristjansson ◽

Olafur Th. Magnusson ◽

Haflidi M. Gudmundsson ◽

Gudni A. Alfredsson ◽

Hiroshi Matsuzawa

Keyword(s):

Vibrio Species ◽

Proteinase K ◽

Species Comparison

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Release of Pharmaceutical Peptides in an Aggregated State: Using Fibrillar Polymorphism to Modulate Release Levels

Colloids and Interfaces ◽

10.3390/colloids3010042 ◽

2019 ◽

Vol 3 (1) ◽

pp. 42 ◽

Cited By ~ 3

Author(s):

Jens Madsen ◽

Gunna Christiansen ◽

Lise Giehm ◽

Daniel Otzen

Keyword(s):

Control Release ◽

Proteinase K ◽

Sustained Delivery ◽

Soluble Peptide ◽

Surfactant Aggregates ◽

Dodecyl Maltoside ◽

Multiple Cycles ◽

The Stability ◽

Traditional Approaches ◽

Release Profiles

Traditional approaches to achieve sustained delivery of pharmaceutical peptides traditionally use co-excipients (e.g., microspheres and hydrogels). Here, we investigate the release of an amyloidogenic glucagon analogue (3474) from an aggregated state and the influence of surfactants on this process. The formulation of peptide 3474 in dodecyl maltoside (DDM), rhamnolipid (RL), and sophorolipid (SL) led to faster fibrillation. When the aggregates were subjected to multiple cycles of release by repeated resuspension in fresh buffer, the kinetics of the release of soluble peptide 3474 from different surfactant aggregates all followed a simple exponential decay fit, with half-lives of 5–18 min and relatively constant levels of release in each cycle. However, different amounts of peptide are released from different aggregates, ranging from 0.015 mg/mL (3475-buffer) up to 0.03 mg/mL (3474-DDM), with 3474-buffer and 3474-RL in between. In addition to higher release levels, 3474-DDM aggregates showed a different amyloid FTIR structure, compared to 3474-RL and 3474-SL aggregates and a faster rate of degradation by proteinase K. This demonstrates that the stability of organized peptide aggregates can be modulated to achieve differences in release of soluble peptides, thus coupling aggregate polymorphism to differential release profiles. We achieved aggregate polymorphism by the addition of different surfactants, but polymorphism may also be reached through other approaches, including different excipients as well as changes in pH and salinity, providing a versatile handle to control release profiles.

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Biomineralization improves the stability of a Streptococcus pneumoniae protein vaccine at high temperatures

Nanomedicine ◽

10.2217/nnm-2021-0023 ◽

2021 ◽

Author(s):

Yajun Min ◽

Wenchun Xu ◽

Yunju Xiao ◽

Jiangming Xiao ◽

Zhaoche Shu ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

High Temperatures ◽

Vaccine Development ◽

Effective Means ◽

Proteinase K ◽

Protein Vaccine ◽

Protein Vaccines ◽

The Stability

Aim: Protein vaccines have been the focus of research for vaccine development due to their safety record and facile production. Improving the stability of proteins is of great significance to the application of protein vaccines. Materials & methods: Based on the proteins pneumolysin and DnaJ of Streptococcus pneumoniae, biomineralization was carried out to prepare protein nanoparticles, and their thermal stability was tested both in vivo and in vitro. Results: Mineralized nanoparticles were formed successfully and these calcium phosphate-encapsulated proteins were resistant to proteinase K degradation and were thermally stable at high temperatures. The mineralized proteins retained the immunoreactivity of the original proteins. Conclusion: Mineralization technology is an effective means to stabilize protein vaccines, presenting a safe and economical method for vaccine administration.

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Enzyme-instructed morphological transition of the supramolecular assemblies of branched peptides

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.16.221 ◽

2020 ◽

Vol 16 ◽

pp. 2709-2718

Author(s):

Dongsik Yang ◽

Hongjian He ◽

Bing Xu

Keyword(s):

Enzymatic Reaction ◽

Proteolytic Cleavage ◽

Supramolecular Assemblies ◽

Lysine Residue ◽

Proteinase K ◽

Morphological Transition ◽

Cellular Environment ◽

Branched Peptides ◽

The Stability

Here, we report the use of an enzymatic reaction to cleave the branch off branched peptides for inducing the morphological transition of the assemblies of the peptides. The attachment of DEDDDLLI sequences to the ε-amine of the lysine residue of a tetrapeptide produces branched peptides that form micelles. Upon the proteolytic cleavage of the branch, catalyzed by proteinase K, the micelles turn into nanofibers. We also found that the acetylation of the N-terminal of the branch increased the stability of the branched peptides. Moreover, these branched peptides facilitate the delivery of the proteins into cells. This work contributes insights for the development of peptide supramolecular assemblies via enzymatic noncovalent synthesis in cellular environment.

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Position-dependent impact of hexafluoroleucine and trifluoroisoleucine on protease digestion

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.13.279 ◽

2017 ◽

Vol 13 ◽

pp. 2869-2882 ◽

Cited By ~ 7

Author(s):

Susanne Huhmann ◽

Anne-Katrin Stegemann ◽

Kristin Folmert ◽

Damian Klemczak ◽

Johann Moschner ◽

...

Keyword(s):

Amino Acids ◽

Systematic Investigation ◽

Proteinase K ◽

Side Chain ◽

Enzyme Binding ◽

Fluorinated Amino Acids ◽

Rp Hplc ◽

Protease Digestion ◽

Proteolytic Stability ◽

The Stability

Rapid digestion by proteases limits the application of peptides as therapeutics. One strategy to increase the proteolytic stability of peptides is the modification with fluorinated amino acids. This study presents a systematic investigation of the effects of fluorinated leucine and isoleucine derivatives on the proteolytic stability of a peptide that was designed to comprise substrate specificities of different proteases. Therefore, leucine, isoleucine, and their side-chain fluorinated variants were site-specifically incorporated at different positions of this peptide resulting in a library of 13 distinct peptides. The stability of these peptides towards proteolysis by α-chymotrypsin, pepsin, proteinase K, and elastase was studied, and this process was followed by an FL-RP-HPLC assay in combination with mass spectrometry. In a few cases, we observed an exceptional increase in proteolytic stability upon introduction of the fluorine substituents. The opposite phenomenon was observed in other cases, and this may be explained by specific interactions of fluorinated residues with the respective enzyme binding sites. Noteworthy is that 5,5,5-trifluoroisoleucine is able to significantly protect peptides from proteolysis by all enzymes included in this study when positioned N-terminal to the cleavage site. These results provide valuable information for the application of fluorinated amino acids in the design of proteolytically stable peptide-based pharmaceuticals.

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Green synthesis of zinc oxide nanoparticles and their effect on the stability and activity of proteinase K

RSC Advances ◽

10.1039/c5ra24862k ◽

2016 ◽

Vol 6 (48) ◽

pp. 42313-42323 ◽

Cited By ~ 45

Author(s):

Mansoore Hosseini Koupaei ◽

Behzad Shareghi ◽

Ali Akbar Saboury ◽

Fateme Davar ◽

Aboulfazl Semnani ◽

...

Keyword(s):

Zinc Oxide ◽

Green Synthesis ◽

Zinc Oxide Nanoparticles ◽

Environmentally Benign ◽

Proteinase K ◽

Oxide Nanoparticles ◽

Biological Applications ◽

The Stability

The use of environmentally benign materials for the synthesis of zinc oxide nanoparticles offers numerous benefits of eco-friendliness and compatibility for pharmaceutical, biotechnological and biological applications.

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Energy-Dependent Conformational Change in the TolA Protein ofEscherichia coli Involves Its N-Terminal Domain, TolQ, and TolR

Journal of Bacteriology ◽

10.1128/jb.183.14.4110-4114.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4110-4114 ◽

Cited By ~ 59

Author(s):

Pierre Germon ◽

Marie-Céline Ray ◽

Anne Vianney ◽

Jean Claude Lazzaroni

Keyword(s):

Outer Membrane ◽

Parental Strain ◽

Potential Role ◽

Limited Proteolysis ◽

Proteinase K ◽

Terminal Domain ◽

Dependent Change ◽

The Stability ◽

Energy Dependent

ABSTRACT TolQ, TolR, and TolA inner membrane proteins of Escherichia coli are involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in ΔtolQ, ΔtolR, and tolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such as lpp, pal, and Δrfa mutants: the behavior of TolA inlpp mutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the Δrfa mutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.

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Fibrillin assembly: dimer formation mediated by amino-terminal sequences

Journal of Cell Science ◽

10.1242/jcs.112.20.3549 ◽

1999 ◽

Vol 112 (20) ◽

pp. 3549-3558 ◽

Cited By ~ 1

Author(s):

J.L. Ashworth ◽

V. Kelly ◽

R. Wilson ◽

C.A. Shuttleworth ◽

C.M. Kielty

Keyword(s):

Proteinase K ◽

In Vitro Translation ◽

Translation System ◽

Dimer Formation ◽

Amino Terminal ◽

Reducing Conditions ◽

Sds Page ◽

Fibrillin 1 ◽

The Stability

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9–11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.

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