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The Modification of Cystine — Cleavage of Disulfide Bonds
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
There are several approaches to the cleavage of disulfide bonds in proteins. The majority of studies involve the cleavage of the disulfide bond of cystine to the free thiol group of cysteine by reduction. Reduction has been generally accomplished with a mild reducing agent such as β-mercaptoethanol or cysteine. Gorin and co-workers1 have examined the rate of reaction of lysozyme with various thiols. At pH 10.0 (0.025 M borate), the relative rates of reaction were β-mercaptoethanol (2-mercaptoethanol), 0.2; dithiothreitol, 1.0; 3-mercaptopropionate, 0.4; and 2-aminoethanol, 0.01. The results with aminoethanethiol were somewhat surprising since the reaction (disulfide exchange) involves the thiolate anion and 2-aminoethanethiol would be more extensively ionized than the other mercaptans. Dithiothreitol has been a useful reagent in the reduction of disulfide bonds in proteins2 as introduced by Cleland. Dithiothreitol and the isomeric form, dithioerythritol, are each capable of the quantitative reduction of disulfide bonds in proteins. Furthermore, the oxidized form of dithiothreitol has an absorbance maximum at 283 nm (Δϵ = 273) which can be used to determine the extent of disulfide bond cleavage.2 The UV spectra of dithiothreitol and oxidized dithiothreitol are shown in Figure 1. Insolubilized dihydrolipoic acid has also been proposed for use in the quantitative reduction of disulfide bonds.4
Purification of Bovine Milk Xanthine Oxidase
Published in Robert A. Greenwald, CRC Handbook of Methods for Oxygen Radical Research, 2018
Massey et al.5 and Hart et al.6 purified milk xanthine oxidase with and without pancreatin treatment and observed no spectral or catalytic differences between the two preparations. As a result, most studies on milk xanthine oxidase have been performed with pancreatin-treated enzyme. The first indication that the two methods yield products with significant functional differences was furnished by Battelli et al.,7 who showed that unproteolyzed xanthine oxidase but not the proteolytically treated enzyme could be converted into an NAD+ -dependent dehydrogenase by dithioerythritol. Treatment with chymotrypsin was also shown to alter the electrophoretic mobility of the enzyme on polyacrylamide gels. Waud et al.8 devised a procedure for purification of the milk enzyme in an undegraded form and showed that unproteolyzed enzyme differed from proteolytically prepared enzyme in native molecular weight, subunit structure, and electrophoretic mobility.
The Structure of Pyruvate Carboxylase
Published in D. B. Keech, J. C. Wallace, Pyruvate Carboxylase, 2018
John C. Wallace, Simon B. Easterbrook-Smith
Although various chromatographic purifications had been attempted previously (with only rather disappointing results), the key to the success of the DEAE-Sephadex-A50 procedure introduced by Scrutton and Fung749 was their unconventional use of a linear gradient of increasing ammonium sulfate concentration in a phosphate-buffered medium. Providing a reducing agent (e.g., dithioerythritol [0.1 mM]) is present in all buffers, this step regularly achieves a 10 to 20-fold purification with a 60 to 70% recovery of enzymic activity. In those situations where there is a particularly high ratio of glutamate dehydrogenase to pyruvate carboxylase activity (e.g., in ruminant liver), an improved separation of these two enzymes can be achieved during this anion-exchange chromatography by combining a pH gradient along with the ionic strength gradient, as described by Goss et al.328
The inhibitory effects of pimozide, an antipsychotic drug, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells
Published in Drug and Chemical Toxicology, 2023
Mi Seon Seo, Jin Ryeol An, Ryeon Heo, Minji Kang, Seojin Park, Seo-Yeong Mun, Hongzoo Park, Eun-Taek Han, Jin-Hee Han, Wanjoo Chun, Geehyun Song, Won Sun Park
The rabbits were anesthetized with an intravenous injection of sodium pentobarbitone (35 mg/kg) and heparin (125 U/kg). The hearts were immediately removed from the euthanized rabbits and immersed in an ice-cold NT solution. The coronary arteries were gently separated from the left ventricle and perivascular connective tissue was trimmed off under a stereomicroscope. The coronary arteries were cut open along longitudinal axes, and their endothelial cells were removed by injecting an air bubble. The smooth muscle cells were isolated by two-step enzymatic digestion. First, arterial tissues were digested in 1 mL Ca2+-free NT solution containing papain (1.6 mg/mL), bovine serum albumin (1.4 mg/mL), and dithioerythritol (1.4 mg/mL) in a 37 °C water bath for 25 min. The tissue was rinsed and digestion was continued in a second digestion solution containing 2.5 mg/mL collagenase instead of papain for 18 min. After enzymatic digestion, a cell suspension was obtained by agitating the tissue using a heat-polished Pasteur pipette in KB solution. The cells obtained were stored at 4 °C and used for electrophysiological experiments within 6 h.
Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue
Published in Scandinavian Cardiovascular Journal, 2018
Ruha Cerrato, Mark Crabtree, Charalambos Antoniades, Karolina Kublickiene, Ernesto L. Schiffrin, Keith M. Channon, Felix Böhm
sEnd.1 cells [9] were investigated first due to their large content of BH4 and a possible ET-induced reduction would therefore be readibly detectable. sEnd.1 cells and HUVEC were grown to sub-confluence and then incubated with or without ET-1 (0.1nM to 0.1 μM) in combination with the dual ETA/ETB receptor antagonist bosentan (3 μM) for various time points. The experiments with HUVEC were repeated five times to ensure validity of the data. The cells were harvested by trypsinization or scraping. Cell pellets were re-suspended in phosphate-buffered saline (50 mM), pH 7.4, containing dithioerythritol (DTE; 1 mM) and EDTA (100 μM) and subjected to three freeze-thaw cycles. After centrifugation (15 min at 13,000 rpm and 4 °C), samples were transferred to new, cooled micro tubes and precipitated with cold phosphoric acid (1 M), trichloroacetic acid (2 M) and DTE (1 mM). Samples were vigorously mixed and then centrifuged for 15 min at 13,000 rpm and 4 °C. Samples were injected onto an isocratic HPLC system.
Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor
Published in OncoImmunology, 2018
Silvia Gaggero, Maurizio Bruschi, Andrea Petretto, Monica Parodi, Genny Del Zotto, Chiara Lavarello, Carola Prato, Laura Santucci, Alessandra Barbuto, Cristina Bottino, Giovanni Candiano, Alessandro Moretta, Massimo Vitale, Lorenzo Moretta, Claudia Cantoni
Concentrated HEK293T-SN and HEK293T-SN-biot (300 μg for Western blot and 600 μg for preparative gels) were solubilized in the reduction/alkylation solution containing 8 M urea, 4% CHAPS, 5 mM tributylphosphine (TBP), 20 mM iodoacetamide (IAA), 40 mM Tris, and 0.1 mM EDTA for 1 h. To prevent over-alkylation during the isoelectro focusing (IEF) step, excess of IAA was neutralized by adding an equimolar amount of DTT. Finally, samples were dissolved in the focusing/re-hydration solution, i.e. 7 M urea, 2 M thiourea, 4% CHAPS, and 15 mM dithioerythritol (DTE) and a 0.6% (v/v) carrier ampholyte cocktail, containing 40% of the pH 3.5–10 and 60% of the pH 4–8 intervals (BDH Biochemical, 44430 2F) and loaded onto home-made non-linear pH 3–10 strips.72 After IEF runs, the strips were equilibrated in 6 M Urea, 50 mM Tris-HCl pH 8.8, 2% (w/v) SDS, 30% (v/v) glycerol, and traces of bromophenol blue; the proteins were separated using a SDS-PAGE (T% 8–16) and transferred onto nitrocellulose membranes (Protran BA85, Whatman, 10402588) with a semidry system. The membranes were saturated with 3% w/v polyvinyl-pyrrolidone (PVP) in TBS and incubated overnight separately with NKp44Fc, NKp30Fc, or NKp46Fc in 3% w/v BSA in TBS-Tween 0.15% v/v (TBS-T). Membranes were then rinsed in TBS-T and incubated with anti-human IgG HRP-conjugated mAb. HEK293T-SN-biot was subjected to the same procedure and immunoblotted with Neutravidin-HRP (ThermoFisher, 31001). For preparative experiments, SDS-gels were stained with “blue silver” colloidal Coomassie.73 Images were digitalized using ChemiDoc Touch (Bio-Rad) and analyzed with PDQuest software (Bio-Rad).