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Glutamate Dehydrogenase
Published in Elling Kvamme, Glutamine and Glutamate in Mammals, 1988
Thus, under these conditions the enzyme has a hexameric structure and this has been confirmed by electron microscopic examination52 which indicated the subunits to be arranged in two layers of trimers in the form of a triangular antiprism.48,52 The hexameric structure has also been confirmed by cross-linking studies.53 Studies on the denaturation of the enzyme in the presence of guanidinium chloride have shown that dissociation into trimers first occurs in a process that involves little loss of conformation, as assessed by circular dichroism, followed by complete dissociation into monomers associated with unfolding of the polypeptide chains.54,55 Studies on the effects of dilution of the guanidinium chloride-treated enzyme have shown that the trimers are without catalytic activity but they can reassociate to form the active hexamer. In contrast the monomeric form produced at higher guanidinium chloride concentrations cannot be renatured.44 The technique of irradiation-inactivation has also indicated the smallest enzymically active form of glutamate dehydrogenase to be the hexamer.56,57
Nonhistone Proteins in Androgen Action
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
Saline-soluble nuclear proteins also contain a basic protein acceptor(s). Mainwaring et al.53 prepared nuclear proteins from castrated rat prostate by extraction with 2 M NaCl and fractionation by chromatography on Bio-Rex®-70 into a nonadsorbed fraction and fractions eluted successively with 0.4 M NaCl and 10% guanidinium chloride. These nuclear fractions were each coupled to CNBr-activated Sepharose® 2B and assayed for binding of [3H]DHT-receptor preparation in the presence and absence of 0.5 M KCl. Only the fraction eluted by guanidinium chloride bound specifically to the receptor complex. Liquid isoelectric focusing of the nuclear extract showed that the high-affinity binding nuclear proteins had pI of 8.6 to 9.3, indicating they are basic proteins. The low-affinity binding nuclear proteins were acidic proteins with pI 3 to 7. Fractionation of the nuclear extract either by sucrose gradient centrifugation or by gel filtration on Sephadex® G-75 showed that the high affinity acceptor proteins had an average sedimentation coefficient of 6.5 S and a molecular weight of 70,000. By comparison, the low-affinity nuclear binding proteins were 1.5 to 5.5 S proteins. These results are at variance with the soluble acceptor preparations described previously which apparently are acidic proteins and, at least in one case, of low molecular weight. The basic acceptor could be localized in the 0.4 to 2.0 M KCl fraction. These results show that the saline-soluble nuclear proteins contain more than one acceptor.
Ceruloplasmin
Published in René Lontie, Copper Proteins and Copper Enzymes, 1984
Since a difference in subunit structure of Cp isolated from different mammalian species would be interesting, Rydén79 undertook a comparative study using gel filtration in 6 M guanidinium chloride of reduced and alkylated proteins.78 Under these conditions a direct measurement of polypeptide chain length is obtained. Protein from fresh pig, horse, and rabbit sera were all found to possess a single peptide chain with a Mr close to 120,000.79 A sample of human Cp prepared from fresh serum was subsequently found to contain a peptide chain of the same size. In order to check the possibility that the commonly used Cp preparations do indeed contain proteolytic fragments, Rydén prepared a single-chain sample from fresh serum in the presence of a proteinase inhibitor and treated it with trypsin for short periods. Fragments similar to the previously described “subunits” were obtained.33 Rydén’s results have subsequently been confirmed and extended by Løwenstein80 using immunological methods, and by Kingston et al.36 and Moshkov et al.35 using SDS-gel electrophoresis.
The impact of concurrent training and antioxidant supplementation on the factors associated with the ocular lens opacity in diabetic rats
Published in Archives of Physiology and Biochemistry, 2022
Mousa Amirahmadi, Mohsen Salesi, Reza Yousefi, Farhad Daryanosh, Javad Nemati, Boris I. Kurganov
The carbonyl contents of the lens proteins were determined in the presence of 2,4-dinitrophenylhydrazine (DNPH) using a slight modification (Levine 1994). In brief, TSPs were precipitated with 20% (v/v) ice-cold TCA and incubated at 4 °C for a period of 10 min. The precipitates were then centrifuged at 11,000 g for 3 min and the protein pellet was resuspended in 10 mM DNPH (2 N) HCl (0.5 mL). The protein samples were subjected to the continuous vortex for 1 h at room temperature. The protein precipitation was then performed with 0.5 mL of 20% (v/v) TCA followed by centrifugation at 11,000 g for 3 min. The pellet was washed with 1 mL ethanol–ethyl acetic acid mixture (1:1v/v) to remove the excess reagents. The protein samples which incubated for 10 min at room temperature were subjected to the centrifugation at 11,000 g for 5 min. The supernatant was discarded and the pellet was washed twice with the ethanol–ethyl acetic acid mixture. The protein pellet was finally suspended in 1 mL guanidinium chloride (GdnHCl) (6 M) which was already dissolved in HCl (2 N). Subsequently, the samples were incubated at 37 °C for 15–30 min to achieve complete solubility of the proteins. All protein samples were then centrifuged to remove the insoluble materials. The absorbance of protein samples was read at 360 nm against GdnHCl (6 M) as the blank solution. The carbonyl content was then determined using a molar extinction coefficient of 22,000 M−1 cm−1 and expressed as nmol/mg protein (Moghadam et al. 2017).
Nanobodies and Cancer: Current Status and New Perspectives
Published in Cancer Investigation, 2018
Alessandro Allegra, Vanessa Innao, Demetrio Gerace, Doriana Vaddinelli, Andrea Gaetano Allegra, Caterina Musolino
The cost-effective creation of Nbs is a relevant benefit compared with the high fabrication costs of traditional mAbs. However, when trivalent or other multivalent scaffolds are made, production in mammalian cells is necessary and costs will rise. The monomeric character and elevated aqueous solubility participate in the cost effective creation of Nbs in a multiplicity of cell expression systems. Nbs have low predisposition for aggregation and are extremely stable (79–82). They preserve full antigen-binding ability in harsh thermal (60–80°C) and chemical (2–3 M guanidinium chloride) denaturing conditions (83). Moreover, the small dimension proteins can resist proteolytic degradation in the gastrointestinal tract, which theoretically allows oral administration of Nbs (84).
Guanidine-based disinfectants, polyhexamethylene guanidine-phosphate (PHMG-P), polyhexamethylene biguanide (PHMB), and oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) induced epithelial-mesenchymal transition in A549 alveolar epithelial cells
Published in Inhalation Toxicology, 2019
Yong Joo Park, Mi Ho Jeong, In Jae Bang, Ha Ryong Kim, Kyu Hyuck Chung
In this study, we investigated the cellular damage and fibrotic response induced by PHMG-P, polyhexamethylene biguanide (PHMB), and oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH), which are guanidine-based polymers. It is necessary to evaluate the common toxicity mechanisms of guanidine-based polymers to predict toxic effects owing to the continued development and use of many new guanidine-based polymers. We measured the cytotoxicity and plasma membrane disruption of PHMG-P, PHMB, and PGH in human alveolar epithelial cells and evaluated the induction of EMT.