Selective reduction of a disulphide bridge in hen ovotransferrin.
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ABSTRACT:
SUBMITTER: Williams J
PROVIDER: S-EPMC1145035 | biostudies-other | 1985 Jun
REPOSITORIES: biostudies-other
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Project description:1. The distribution of iron between the two iron-binding sites in partially saturated ovotransferrin was studied by labelling with 55Fe and 59Fe and by gel electrophoresis in a urea-containing buffer. 2. When iron is added in the form of chelate complexes at alkaline pH, binding occurs preferentially at the N-terminal binding site. In acid, binding occurs preferentially at the C-terminal site. 3. When simple iron donors (ferric and ferrous salts) are used the metal is distributed at random between the binding sites, as judged by the gel-electrophoresis method. The double-isotope method shows a preference of ferrous salts for the N-terminal site. 4. Quantitative treatment of the results of double-isotope labelling suggests that in the binding of iron to ovotransferrin at alkaline pH co-operative interactions between the sites occur. These interactions are apparently absent in the displacement of copper and in the binding of iron at acid pH.
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Project description:Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer's disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.
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Project description:Acute Hepatopancreatic Necrosis Disease (AHPND), a highly destructive shrimp disease, has inflicted severe setbacks on the shrimp farming industry worldwide. As the use of antibiotics is discouraged due to emerging antibiotic-resistant bacteria and the pollution of ecosystems, there is a pressing demand for novel, sustainable alternatives. Hence, the influence of bovine lactoferrin (bLF) and hen ovotransferrin (OT), two natural antimicrobial proteins, on the growth of three AHPND-causing Vibrio parahaemolyticus (Vp) strains (M0904, TW01 and PV1) was examined. Additionally, we explored their potential to affect selected Vp virulence factors such as biofilm formation, swimming and swarming, cell surface hydrophobicity, and activity of released lipases and caseinases. Lag phases of all bacterial growth curves were significantly prolonged in the presence of bLF or OT (1, 5 and 10 mg/mL), and bLF (5 and 10 mg/mL) completely inhibited growth of all strains. In addition, bLF or OT significantly reduced biofilm formation (all tested bLF and OT concentrations for Vp M0904 and Vp PV1), bacterial swimming motility (0.5 mg/mL bLF and OT for Vp M0904 and Vp TW01; 1 mg/mL bLF and OT for all strains), cell surface hydrophobicity (for all strains, all bLF and OT concentrations tested except for 0.125 mg/mL OT for Vp PV1) and lipase activity (1 mg/mL bLF and OT for all strains and 0.5 mg/mL bLF and OT for Vp PV1). These promising in vitro results suggest that bLF and/or OT might be used as novel agents for combating AHPND and warrant further research to elucidate the underlying mechanisms of action to fully unlock their potential for AHPND disease management.
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Project description:1. When iron-saturated hen ovotransferrin was treated with subtilisin the N-terminal half was digested at a faster rate than the C-terminal half, allowing the latter to be isolated as a single-chain fragment of mol.wt 35000. 2. In mildly acid conditions iron-ovotransferrin loses iron preferentially from its N-terminal binding site. Trypsin digestion of the resulting monoferric ovotransferrin also gave rise to a C-terminal fragment. 3. Comparison of the N-terminal fragment with the C-terminal fragments shows differences in composition, peptide 'maps', CNBr-cleavage patterns and antigenic structures. The C-terminal fragments carry the carbohydrate group of ovotransferrin. 4. Both N-terminal and C-terminal fragments donate their bound iron to rabbit reticulocytes.
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Project description:1. Iron was added to hen ovotransferrin to 30% saturation and the protein was digested with trypsin or chymotrypsin. 2. Iron-binding fragments were isolated. They carried one atom of iron/mol (mol.wt. 35000) and consisted of a single polypeptide chain derived from the N-terminal half of the protein. Carbohydrate was not present. 3. The fragments were able to bind a variety of metals and to donate iron to reticulocytes.
| S-EPMC1168181 | biostudies-other
Project description:1. Hen ovotransferrin was treated with CNBr and fractionated by gel filtration. 2. After further treatment by reduction and carboxymethylation a carbohydrate-containing fragment of molecular weight 11990 was obtained (fragment BCd). 3. The amino acid sequence of this fragment was determined. It consists of a single chain of 94 residues. 4. The structure of a tryptic glycopeptide derived from whole ovotransferrin permitted a further eight residues to be assigned at the N-terminus of fragment BCd. 5. Heterogeneity was found at two positions. 6. Further evidence has been deposited as Supplementary Publication SUP 50045 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1975), 145, 5.
| S-EPMC1165473 | biostudies-other
Project description:1. Glycopeptides were prepared from proteolytic digests of ovotransferrin and serum transferrin of the hen. The carbohydrate compositions and amino acid sequences of the peptides were studied. 2. The bulk of the carbohydrate of ovotransferrin is present as a single oligosaccharide composed of 4 residues of mannose and 8 residues of N-acetylglucosamine. Transferrin has most of its carbohydrate in a single unit composed of 2 residues of mannose, 2 residues of galactose, 3 residues of N-acetylglucosamine and either 1 or 2 residues of sialic acid. 3. The amino acid sequences of the glycopeptides carrying these different oligosaccharides are the same in ovotransferrin and serum transferrin, showing that the carbohydrate groups are attached to the same site on the protein molecule.
| S-EPMC1198769 | biostudies-other
Project description:The kinetics of iron and copper binding to hen's-egg apo-ovotransferrin were studied by using citrate chelates of these metals at pH9.3 in borate buffer in the presence of bicarbonate. The kinetics of the absorbance change associated with the formation of the final product show a fast process, which is pseudo-first-order, where the reagents are in excess with respect to the protein, and the citrate concentration is higher than 25mM. At lower citrate concentration, the progress curves are clearly biphasic. There is marked dependence of the rate of the reaction on bicarbonate concentration, which may be interpreted as a displacement reaction of the ligand-metal-protein ternary complex. The kinetics have been interpreted in the framework of a reaction scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent colour development by displacement of the chelator by bicarbonate. The pH-dependence of this reaction supports the belief that tyrosine residues are involved in the process of iron-binding. The overall similarity of kinetics for iron and copper binding, notwithstanding their different co-ordination preferences, suggests that the process of metal-binding or chromophore development for the two metal complexes must be similar.
| S-EPMC1165463 | biostudies-other
Project description:1. The thiol and disulphide contents of hen ovalbumin were investigated by p-chloromercuribenzoate titration, by determination of cysteic acid content after performic acid oxidation, by measurement of uptake of radioactive iodoacetic acid, and by assay of S-aminoethylcysteine after reaction with ethyleneimine. All results showed that ovalbumin had 6 half-cystine residues. Experiments with and without reducing agents demonstrated that there were 4 thiol groups and 1 disulphide bond. 2. A peptide containing equimolar amounts of S-carboxymethyl-cysteine, serine, valine and proline, but no lysine or arginine, was obtained by radioactive labelling of the cysteine residues with iodo[(14)C]acetic acid followed by electrophoretic and chromatographic separation of tryptic digests. It was concluded that the C-terminal sequence of ovalbumin is -Cys-Val-Ser-Pro. 3. The location of the disulphide bond was studied by using a double-labelling technique. It was shown that one end of the disulphide was located in this C-terminal peptide.
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Project description:As a feed additive, xylanase has been widely applied in the feed of monogastric animals, which contains multiple plant polysaccharides. However, during feed manufacture, the high pelleting temperatures challenge wild-type xylanases. The aim of this study was to improve the thermostability of Aspergillus sulphureus acidic xylanase. According to the predicted protein structure, a series of disulphide bridges and proline substitutions were created in the xylanase by PCR, and the mutants were expressed in Pichia pastoris. Enzyme properties were evaluated following chromatographic purification. All the recombinant enzymes showed optima at pH 3.0 and 50 °C or 55 °C and better resistance to some chemicals except for CuSO4. The specific activity of the xylanase was decreased by introduction of the mutations. Compared to the wild-type enzyme, a combined mutant, T53C-T142C/T46P, with a disulphide bond at 53-142 and a proline substitution at 46, showed a 22-fold increase of half-life at 60 °C. In a 10-L fermentor, the maximal xylanase activity of T53C-T142C/T46P reached 1,684 U/mL. It was suggested that the T53C-T142C/T46P mutant xylanase had excellent thermostability characteristics and could be a prospective additive in feed manufacture.
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