Detection of a Myc-associated protein by chemical cross-linking

1989 ◽  
Vol 9 (2) ◽  
pp. 865-868
Author(s):  
D A Gillespie ◽  
R N Eisenman
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Nuclear Protein ◽  
Cross Linking ◽  
Cross Linker ◽  
Chemical Cross Linking

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

scholarly journals Detection of a Myc-associated protein by chemical cross-linking.

1989 ◽  
Vol 9 (2) ◽  
pp. 865-868 ◽  
Author(s):  
D A Gillespie ◽  
R N Eisenman
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Nuclear Protein ◽  
Cross Linking ◽  
Cross Linker ◽  
Chemical Cross Linking

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

scholarly journals Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains

2020 ◽  
Author(s):  
Jan Fiala ◽  
Zdeněk Kukačka ◽  
Petr Novák
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Water Soluble ◽  
Cross Linking ◽  
Side Chains ◽  
Model Protein ◽  
Cross Linker ◽  
Progressive Technology ◽  
The Impact ◽  
Chemical Cross Linking

The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.

scholarly journals Gelatin-Based Hydrogels through Homobifunctional Triazolinediones Targeting Tyrosine Residues

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 589 ◽  
Author(s):  
Roberto Guizzardi ◽  
Luca Vaghi ◽  
Marcello Marelli ◽  
Antonino Natalello ◽  
Ivan Andreosso ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Drug Delivery Systems ◽  
Chemical Nature ◽  
Mechanical Stability ◽  
Cross Linking ◽  
Swelling Properties ◽  
Tyrosine Residues ◽  
Cross Linker ◽  
The Cross ◽  
Chemical Cross Linking

Gelatin is a biopolymer with interesting properties that can be useful for biomaterial design for different applications such as drug delivery systems, or 3D scaffolds for tissue engineering. However, gelatin suffers from poor mechanical stability at physiological temperature, hence methods for improving its properties are highly desirable. In the present work, a new chemical cross-linking strategy based on triazolinedione ene-type chemistry towards stable hydrogel is proposed. Two different homobifunctional 1,2,4-triazoline-3,5(4H)-diones, namely 4,4′-hexane-1,6-diylbis(3H-1,2,4-triazoline-3,5(4H)-dione) 1 and 4,4′-[methylenebis(4,1-phenylene)]bis(3H-1,2,4-triazoline-3,5(4H)-dione) 2 were used as cross-linkers in different ratio to tyrosine residues in gelatin. The reaction was proved effective in all experimented conditions and hydrogels featured with different thermal stability were obtained. In general, the higher the cross-linker/tyrosine ratio, the more thermostable the hydrogel. The swelling properties are strictly dependent upon the chemical nature of the cross-linker.

scholarly journals A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

1984 ◽  
Vol 99 (1) ◽  
pp. 188-198 ◽  
Author(s):  
N G Kravit ◽  
C S Regula ◽  
R D Berlin
Keyword(s):  
Molecular Weight ◽  
Room Temperature ◽  
Molecular Form ◽  
Cross Linking ◽  
Electrophoretic Patterns ◽  
Minority Species ◽  
Associated Proteins ◽  
Dimethyl Suberimidate ◽  
Quantitative Examination ◽  
Chemical Cross Linking

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry

2018 ◽  
Vol 115 (44) ◽  
pp. 11162-11167 ◽  
Author(s):  
Bing Yang ◽  
Haifan Wu ◽  
Paul D. Schnier ◽  
Yansheng Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Current Method ◽  
Thermal Fluctuations ◽  
Limited Range ◽  
Live Cells ◽  
Cross Linking ◽  
Cross Links ◽  
Cross Linker ◽  
Sulfonyl Fluoride ◽  
Chemical Cross Linking

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a “plant-and-cast” cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues “planting” the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then “cast” across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme–substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

scholarly journals The interaction between subunits in the tubulin dimer

1985 ◽  
Vol 230 (2) ◽  
pp. 551-556 ◽  
Author(s):  
L Serrano ◽  
J Avila
Keyword(s):  
Formic Acid ◽  
Limited Proteolysis ◽  
Trypsin Digestion ◽  
Cross Linking ◽  
Β Subunit ◽  
Α Subunit ◽  
Tubulin Dimer ◽  
Cross Linker ◽  
Chemical Cross Linking ◽  
Β Tubulin

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between α- and β-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the α-subunit into two fragments, whereas chymotrypsin cleaved the β-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the α-tubulin N-terminal fragment with β-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.

scholarly journals Some Properties of Chemical Cross-Linking Biohydrogel from Starch and Chitosan

2019 ◽  
Vol 2019 ◽  
pp. 1-6
Author(s):  
Titi Candra Sunarti ◽  
M. Irsan Febrian ◽  
Eka Ruriani ◽  
Indah Yuliasih
Keyword(s):  
Acid Concentration ◽  
Methacrylic Acid ◽  
Industrial Application ◽  
High Capacity ◽  
Monomer Conversion ◽  
Cross Linking ◽  
High Ability ◽  
Grafting Efficiency ◽  
Cross Linker ◽  
Chemical Cross Linking

Chemical cross-linking was developed to prepare starch and chitosan-based hydrogels. First, the precursor of starch was synthesized through the reaction of carboxymethylation with sodium monochloroacetate, and then chitosan was grafted by using methacrylic acid as cross-linker. In this research, sago and cassava starches were used and mixed with chitosan, and the effect of methacrylic acid concentration was investigated to determine the grafting parameters and hydrogel characteristics. Compared to native starch and carboxymethylated starch, hydrogels from both starches have high ability to swell and high capacity to absorb water and oil. The highest grafting yield, grafting efficiency, and monomer conversion were achieved by experiment using 0.550 g of methacrylic acid per g of CMS-chitosan mixture. These hydrogels have a good potency as biodegradable absorbents for pharmaceutical and industrial application.

scholarly journals Evaluation of Low Molecular Weight Cross Linked Chitosan Nanoparticles, to Enhance the Bioavailability of 5-Flourouracil

Dose-Response ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 155932582110253
Author(s):  
Aisha Sethi ◽  
Mahmood Ahmad ◽  
Tayyaba Huma ◽  
Ikrima Khalid ◽  
Imtiaz Ahmad
Keyword(s):  
Drug Delivery ◽  
Molecular Weight ◽  
Drug Release ◽  
Zeta Potential ◽  
Sustained Release ◽  
Drug Delivery Systems ◽  
Chitosan Nanoparticles ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Chemical Cross Linking

The present study aimed to formulate 5-fluorouracil loaded cross linked chitosan nanoparticles based on chemical cross-linking of low molecular weight chitosan with glutaraldehyde by reverse micelles technique as 5-FU is less hydrophobic, relatively potent, has a shorter half-life, is rapidly metabolized, less tolerated, and has low oral bioavailability; therefore, we aimed to formulate potential nanocarriers of 5-FU for efficient drug delivery to specific targeted areas of action, reduce oral toxicity, improve tolerability and therapeutic outcomes of 5-FU, in a restricted fashion to enhance the bioavailability of 5-FU. Nanoparticles were formulated by the reverse micelle method based on the chemical cross-linking of glutaraldehyde (25% aqueous solution) into a w/o emulsion in different ratios. LMWCH-NPs were characterized for post-formulation parameters by mean particle size, zeta potential, %age yield, loading/entrapment efficiency, Fourier transform infrared spectroscopy (FTIR), DSC/TGA, TEM, PXRD, drug release at pH 1.2, and pH 7.4. 5-FU loaded NPs showed a size range (198 nm-200 nm) and zeta potential (−39mV to −41mV), which ensured mechanical stability and increased retention time in blood vessels by the sustained release properties of biodegradable nanocarrier drug delivery systems. % age yield showed the range 92% to 96% while % LC ranged 2.0% to 3.4% and %EE ranged 40% to 43%. The TEM images showed spherical nanoparticles. FTIR revealed the compatibility between the drug and the cross-linked polymer. DSC/TGA ensured the thermal stability of the drug, while the solid-state stability of the drug-loaded cross-linked chitosan nanoparticles was evaluated by powder X-ray diffraction (PXRD) analysis. Drug release studies were performed using the dialysis bag technique at both pH (1.2 and 7.4) to mimic the gastrointestinal tract. Highly stable NPs displayed targeted release in phosphate buffer pH 7.4 at 37°C. Fickian diffusion was the predominant release with an R2 value of 0.9975-0.9973—and an N value 0.45-0.53. Prepared nanoparticles are inert, biodegradable, and biocompatible drug delivery systems for sustained release of 5-FU with maximum therapeutic efficacy and bioavailability.

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