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Methods in Enzymology 2014Antibodies will be immobilized on a cyanogen bromide-activated Sepharose for subsequent use in pull-down assays or immunoaffinity purification.
Antibodies will be immobilized on a cyanogen bromide-activated Sepharose for subsequent use in pull-down assays or immunoaffinity purification.
Topics: Antibodies, Immobilized; Antibodies, Monoclonal; Chromatography, Affinity; Cyanogen Bromide; Resins, Synthetic; Sepharose
PubMed: 24674060
DOI: 10.1016/B978-0-12-420119-4.00003-3 -
The Journal of Biological Chemistry Dec 1979Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid...
Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.
Topics: Amino Acids; Collagen; Cyanogen Bromide; Female; Humans; Macromolecular Substances; Peptide Fragments; Placenta; Pregnancy
PubMed: 500696
DOI: No ID Found -
Molecules (Basel, Switzerland) Feb 2020The influence of buffer type, co-solvent type, and acyl chain length was investigated for the enantioselective hydrolysis of racemic 4-arylbut-3-en-2-yl esters using...
The influence of buffer type, co-solvent type, and acyl chain length was investigated for the enantioselective hydrolysis of racemic 4-arylbut-3-en-2-yl esters using Lecitase Ultra (LU). Immobilized preparations of the Lecitase Ultra enzyme had significantly higher activity and enantioselectivity than the free enzyme, particularly for 4-phenylbut-3-en-2-yl butyrate as the substrate. Moreover, the kinetic resolution with the immobilized enzyme was achieved in a much shorter time (24-48 h). Lecitase Ultra, immobilized on cyanogen bromide-activated agarose, was particularly effective, producing, after 24 h of reaction time in phosphate buffer (pH 7.2) with acetone as co-solvent, both ()-alcohols and unreacted ()-esters with good to excellent enantiomeric excesses (ee 90-99%). These conditions and enzyme were also suitable for the kinetic separation of racemic ()-4-phenylbut-3-en-2-yl butyrate analogs containing methyl substituents on the benzene ring (,), but they did not show any enantioselectivity toward ()-4-(4'-methoxyphenyl)but-3-en-2-yl butyrate ().
Topics: Alcohols; Butyrates; Catalysis; Cyanogen Bromide; Enzymes, Immobilized; Esters; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lipase; Phenylbutyrates; Sepharose; Solvents; Stereoisomerism
PubMed: 32120991
DOI: 10.3390/molecules25051067 -
European Journal of Biochemistry Jul 1982The catabolism of two rabbit globin cyanogen bromide peptides in cell-free extracts of ATP-depleted rabbit reticulocytes has been studied. Proteolysis of the peptides...
The catabolism of two rabbit globin cyanogen bromide peptides in cell-free extracts of ATP-depleted rabbit reticulocytes has been studied. Proteolysis of the peptides (3533 and 5957 molecular weight) proceeded rapidly in the absence of ATP, had a pH optimum of approximately 7.8, and was inhibited by omicron-phenanthroline, N-ethylmaleimide, cystamine zinc and cobalt ions, and puromycin peptide high-molecular-weight aggregates. Proteolysis of puromycin peptides was inhibited by the rabbit globin cyanogen bromide peptides. The ability of cell-free extracts of degrade the globin cyanogen bromide peptides decreased with the reticulocyte maturity. Blocking the globin cyanogen bromide peptide amino groups by succinic and maleic anhydride treatment decreased susceptibility to degradation. It is suggested that the globin cyanogen bromide peptides might provide model substrates, replacing puromycin peptides, for the investigation of ATP-independent proteolytic events.
Topics: Adenosine Triphosphate; Animals; Cell-Free System; Cyanogen Bromide; Globins; Molecular Weight; Peptide Fragments; Rabbits; Reticulocytes
PubMed: 7117254
DOI: 10.1111/j.1432-1033.1982.tb06720.x -
Proceedings of the National Academy of... Sep 1971Tubulin, the subunit protein of microtubules, is a dimer that sediments at 6 S and has a molecular weight of 110,000. Using high resolution polyacrylamide gel...
Tubulin, the subunit protein of microtubules, is a dimer that sediments at 6 S and has a molecular weight of 110,000. Using high resolution polyacrylamide gel electrophoresis, we have demonstrated the presence of two peptide chains, of molecular weight 56,000 and 53,000, in tubulin purified from brain. Two peptide chains of similar molecular weight were identified in each of the A- and B-tubulins isolated from flagella of sea urchin sperm. In all cases, the protein concentrations in the bands were equal. Each of the subunits ran as a single band when eluted from the gel and electrophoresed again in the same type of gel. Chromatography of purified brain tubulin on DEAE-Sephadex columns gave only a single peak containing both subunits in equal amounts. Cyanogen bromide peptides were prepared from each of the bands after elution from polyacrylamide gel. While certain of the peptides appear to be common to both subunits, substantial differences exist between them. The tubulin dimer is composed of two nonidentical subunits.
Topics: Animals; Brain Chemistry; Chromatography, DEAE-Cellulose; Cyanogen Bromide; Electrophoresis, Disc; Isoelectric Focusing; Male; Microtubules; Molecular Weight; Peptides; Proteins; Sea Urchins; Spermatozoa; Swine
PubMed: 5289362
DOI: 10.1073/pnas.68.9.2028 -
Journal of Bacteriology Jan 2000Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-beta-lysine, the first step in lysine degradation in Clostridium subterminale...
Lysine 2,3-aminomutase from Clostridium subterminale SB4: mass spectral characterization of cyanogen bromide-treated peptides and cloning, sequencing, and expression of the gene kamA in Escherichia coli.
Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-beta-lysine, the first step in lysine degradation in Clostridium subterminale SB4. KAM requires S-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5'-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene kamA for C. subterminale SB4 KAM and conditions for its expression in Escherichia coli. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and C. subterminale SB4 chromosomal DNA to produce a portion of kamA containing 1,029 base pairs of the gene. The complete gene was obtained from a genomic library of C. subterminale SB4 chromosomal DNA by use of DNA probe analysis based on the 1,029-base pair fragment. The full-length gene consisted of 1,251 base pairs specifying a protein of 47,030 Da, in reasonable agreement with 47, 173 Da obtained by electrospray mass spectrometry of the purified enzyme. N- and C-terminal amino acid analysis of KAM and its cyanogen bromide peptides firmly correlated its amino acid sequence with the nucleotide sequence of kamA. A survey of bacterial genome databases identified seven homologs with 31 to 72% sequence identity to KAM, none of which were known enzymes. An E. coli expression system consisting of pET 23a(+) plus kamA yielded unsatisfactory expression and bacterial growth. Codon usage in kamA includes the use of AGA for all 29 arginine residues. AGA is rarely used in E. coli, and arginine clusters at positions 4 and 5, 25 and 27, and 134, 135, and 136 apparently compound the barrier to expression. Coexpression of E. coli argU dramatically enhanced both cell growth and expression of KAM. Purified recombinant KAM is equivalent to that purified from C. subterminale SB4.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Clostridium; Cyanogen Bromide; DNA, Bacterial; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Intramolecular Transferases; Mass Spectrometry; Molecular Sequence Data; Peptide Mapping; Peptides; RNA, Transfer, Arg; Recombination, Genetic; Sequence Alignment; Sequence Analysis, DNA
PubMed: 10629195
DOI: 10.1128/JB.182.2.469-476.2000 -
European Journal of Biochemistry Jun 1979Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic...
Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C.
Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3, Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44 000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion.
Topics: Amino Acid Sequence; Carboxypeptidases; Chromatography, Gel; Cyanogen Bromide; Horseradish Peroxidase; Hydrolysis; Peptides; Peroxidases; Pyroglutamyl-Peptidase I; Trypsin
PubMed: 38113
DOI: 10.1111/j.1432-1033.1979.tb13061.x -
Acta Biochimica Polonica 1981D-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from human muscle was S-carboxymethylated, cleaved with cyanogen bromide and citraconylated. The enzymatic...
D-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from human muscle was S-carboxymethylated, cleaved with cyanogen bromide and citraconylated. The enzymatic protein contains 344 amino acid residues, nine of which are methionines. The respective ten cyanogen bromide fragments have been isolated and characterized. Each peptide was purified to homogeneity by Bio-Gel chromatography, high voltage electrophoresis and descending paper chromatography, and characterized by electrochromatography, N-terminal sequence and amino acid composition.
Topics: Chromatography, Gel; Chromatography, Paper; Cyanogen Bromide; Electrophoresis; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Muscles; Peptides
PubMed: 7282214
DOI: No ID Found -
The Journal of Biological Chemistry Jul 1980Cyanogen bromide treatment of reduced, S-carboxymethylated phosphoglycerate kinase yielded 14 major peptides, CNBr-1 (20 residues), CNBr-2 (8 residues), CNBr-3 (33...
Cyanogen bromide treatment of reduced, S-carboxymethylated phosphoglycerate kinase yielded 14 major peptides, CNBr-1 (20 residues), CNBr-2 (8 residues), CNBr-3 (33 residues), CNBr-4 (11 residues), CNBr-5 (104 residues), CNBr-6 (14 residues), CNBr-7 (37 residues), CNBr-8 (7 residues), CNBr-9 (6 residues), CNBr-10 (11 residues), CNBr-11 (19 residues), CNBr-12 (42 residues), CNBr-13 (44 residues), and CNBr-14 (61 residues). The amino acid sequences of all the cyanogen bromide peptides were determined by a combination of automated and manual sequence analysis, and the characterization of tryptic and chymotryptic peptides and peptides obtained by digestion with staphylococcal protease. Two tryptic peptides which were not obtained by direct digestion of whole phosphoglycerate kinase were recovered from cyanogen bromide Peptides CNBr-13 and CNBr-14 and these peptides were purified and sequenced. Based on the information from all the tryptic and cyanogen bromide peptides derived from the enzyme, the proper alignment of these peptides was made. Thus, complete amino acid sequence of human phosphoglycerate kinase consisting of 417 amino acid residues was determined.
Topics: Amino Acid Sequence; Amino Acids; Cyanogen Bromide; Erythrocytes; Humans; Peptides; Phosphoglycerate Kinase; Protein Conformation
PubMed: 7391027
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1981The amino acid sequences of 13 of the 14 cyanogen bromide peptides of horse muscle 3-phosphoglycerate kinase have been determined. These peptides together constitute 75%...
Primary structure of 3-phosphoglycerate kinase from horse muscle. II. Amino acid sequence of cyanogen bromide peptides CB1-CB4 and CB6-CB14, sequence of methionine-containing regions, and complete sequence of the enzyme.
The amino acid sequences of 13 of the 14 cyanogen bromide peptides of horse muscle 3-phosphoglycerate kinase have been determined. These peptides together constitute 75% of the structure of the enzyme. Except for the smallest peptides, automated sequence analysis of the parent peptide was employed together with the automated or manual (5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Edman method) sequencing of relevant peptides obtained by proteolytic digestion. In the case of the five smallest peptides, all between 6 and 11 residues in length, sequences were derived from analyses of the intact peptides. CB14a, a peptide produced in low yield during the preparation of the cyanogen bromide fragments, was helpful in the sequence analysis of CB14. This peptide appeared to have arisen as the result of a cyanogen bromide cleavage on the carboxyl side of a tryptophanyl residue. The sequences of these 13 cyanogen bromide peptides, together with that of CB1, reported previously (Hardy, G. W., Darbre, A., and Merrett, M. (1981) J. Biol. Chem. 256, 10284-10292), have been combined with data obtained from the sequence analysis of tryptic peptides derived from the methionine-containing regions of the enzyme to determine the complete sequence. Isolation of these overlap peptides was facilitated by the labeling of methionyl residues with iodo[2-14C]acetic acid prior to tryptic digestion. Tryptophan-containing tryptic peptides were also isolated, and their sequence analysis enabled the positions of the four tryptophanyl residues to be established. In this way, the problem caused by tryptophan degradation during the preparation of the cyanogen bromide fragments was overcome. The NH2 terminus of CB1, the NH2-terminal cyanogen bromide peptide, was identified as N-acetyl serine by mass spectrometry. The alignment of the cyanogen bromide fragments of horse muscle 3-phosphoglycerate kinase described here is in complete agreement with that based on X-ray analysis of the enzyme reported previously (Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature 279, 773-777). The complete structure is comprised of 416 residues and has a molecular weight of 44,519.
Topics: Amino Acid Sequence; Animals; Autoanalysis; Chymotrypsin; Cyanogen Bromide; Horses; Mass Spectrometry; Muscles; Peptide Fragments; Phosphoglycerate Kinase; Trypsin
PubMed: 7287713
DOI: No ID Found