Isolation and hydroxylysine glycoside content of some cyanogen bromide-cleaved fragments of collagen from bovine corneal stroma.
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ABSTRACT:
SUBMITTER: Panjwani NA
PROVIDER: S-EPMC1184015 | biostudies-other | 1978 Jun
REPOSITORIES: biostudies-other
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Project description:Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.
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Project description:Antibodies will be immobilized on a cyanogen bromide-activated Sepharose for subsequent use in pull-down assays or immunoaffinity purification.
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Project description:1. Procedures are described for the isolation of seven distinct cyanogen bromide fragments in high yield from human serum transferrin. 2. Cyanogen bromide-cleaved transferrin is separated into three fragments (CN-A, CN-B and CN-C) by gel filtration with Sephadex G-100. 3. Four peptides are obtained from CN-A (the largest fragment) after reduction and carboxamidomethylation, by gel filtration in acidic solvents. Two peptides are similarly obtained from fragment CN-B, whereas fragment CN-C is a single cystine-free peptide. 4. The molecular weights of the seven peptides, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, by sedimentation-equilibrium ultracentrifugation and by sequence studies, range from 3100 to 27000. Together they account for a molecular weight of 76200 for transferrin. 5. The two largest fragments contain the carbohydrate attachment sites of the protein, and the smallest fragment is derived from the N-terminus. 6. The amino acid compositions and N-terminal groups of the fragments are reported and the results compared with those of previous investigations.
| S-EPMC1166263 | biostudies-other
Project description:1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.
| S-EPMC1184692 | biostudies-other
Project description:Restoring the anatomical and functional characteristics of the cornea using various biomaterials is especially relevant in the context of a global shortage of donor tissue. Such biomaterials must be biocompatible, strong, and transparent. Here, we report a Viscoll collagen membrane with mechanical and optical properties suitable for replacing damaged stromal tissue. After removing a portion of the stroma, a Viscoll collagen membrane was implanted into the corneas of rabbits. After 6 months, the active migration of host cells into Viscoll collagen membranes was noted, with the preservation of corneal transparency in all experimental animals. Effective integration of the Viscoll collagen membrane with corneal tissue promoted nerve regeneration in vivo, as confirmed by in vivo confocal microscopy. We also demonstrated the safety and efficacy of the Viscoll collagen membrane for corneal stroma regeneration. Thus, in combination with the proposed packaging format that provides long-term storage of up to 10 months, this material has great potential for replacing and regenerating damaged stromal tissues.
| S-EPMC9572218 | biostudies-literature
Project description:1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.
| S-EPMC1184691 | biostudies-other
Project description:The thermal transitions of fibrillar collagen are investigated with second-harmonic generation polarization anisotropy microscopy. Second-harmonic generation images and polarization anisotropy profiles of corneal stroma heated in the 35-80°C range are analyzed by means of a theoretical model that is suitable to probe principal intramolecular and interfibrillar parameters of immediate physiological interest. Our results depict the tissue modification with temperature as the interplay of three destructuration stages at different hierarchical levels of collagen assembly including its tertiary structure and interfibrillar alignment, thus supporting and extending previous findings. This method holds the promise of a quantitative inspection of fundamental biophysical and biochemical processes and may find future applications in real-time and postsurgical functional imaging of collagen-rich tissues subjected to thermal treatments.
| S-EPMC3446693 | biostudies-literature
Project description:Significant amounts of native collagen can be extracted from bovine articular cartilage after removal of the acid mucopolysaccharides by controlled proteolysis. The fraction thus solubilized upon denaturation gives rise to three identical alpha chains. Cleavage of these chains with CNBr generated nine peptides, all of which contain glycine as one-third of their total amino acid residues. Two of the smaller peptides CB-1 and CB-2 contain partially hydroxylated proline. A similar CNBr digest of intact cartilage also gives a series of peptides identical with those obtained from the soluble cartilage collagen. The absence of cross-linking peptides, the fact that only few beta components are seen in articular cartilage collagen and the similarity in peptide pattern between the two collagen fractions investigated, suggests that this collagen is stabilized by a different cross-linking mechanism, possibly involving an association with the tissue proteoglycans.
| S-EPMC1177750 | biostudies-other
Project description:Cyanogn bromide peptides derived from bovine type III collagen and containing reducible cross-links were isolated and identified. Two peptides, alpha 1 (III)CB7 and alpha 1 (III)CB9B, from within the helical portion of the molecule were shown to contain the 'amino donor' residues cross-linked to non-helical 'aldehyde donor' residues in the formation of cross-links. This information, in conjunction with previously published data for the order of the cyanogen bromide peptides [Fietzek, Allman, Rauterberg & Wachter (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 84-86], suggests that in type III collagen intermolecular cross-links are located in the end-overlap regions, so as to stabilize a quarter-stagger arrangement of molecules within the fibre in a similar manner to that proposed for type I and type II collagens.
| S-EPMC1161284 | biostudies-other
Project description:The spatial organization of biofilms is strongly regulated by chemical cues released by settling organisms. However, the exact nature of these interactions and the repertoire of chemical cues and signals that micro-organisms produce and exude in response to the presence of competitors remain largely unexplored. Biofilms dominated by microalgae often show remarkable, yet unexplained fine-scale patchy variation in species composition. Because this occurs even in absence of abiotic heterogeneity, antagonistic interactions might play a key role. Here we show that a marine benthic diatom produces chemical cues that cause chloroplast bleaching, a reduced photosynthetic efficiency, growth inhibition and massive cell death in naturally co-occurring competing microalgae. Using headspace solid phase microextraction (HS-SPME)-GC-MS, we demonstrate that this diatom exudes a diverse mixture of volatile iodinated and brominated metabolites including the natural product cyanogen bromide (BrCN), which exhibits pronounced allelopathic activity. Toxin production is light-dependent with a short BrCN burst after sunrise. BrCN acts as a short-term signal, leading to daily "cleaning" events around the algae. We show that allelopathic effects are H(2)O(2) dependent and link BrCN production to haloperoxidase activity. This strategy is a highly effective means of biofilm control and may provide an explanation for the poorly understood role of volatile halocarbons from marine algae, which contribute significantly to the atmospheric halocarbon budget.
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