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At least 19 recordsLinked to original sources
PubMed1986

Template-directed polymerization of oligoadenylates using cyanogen bromide.

Cyanogen bromide (BrCN) condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aqueous solution. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temperature affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25 degrees C. The coupling yield increased with increase in the poly(U) concentration. Five- to sevenfold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+ and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). N-Cyanoimidazole (1), N,N'-iminodiimidazole (2), and N-carboxamidoimidazole (3) were formed in a reaction of imidazole with BrCN in an aqueous solution. 1 and 2 had much the same condensing activity for the polymerization of adenylates as BrCN. A reaction pathway was proposed in which 1 and 2 are not only intermediates for the production of 3 but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide derivative was detected in a reaction of 5'-AMP with either 1 or 2. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, the phosphorimidazolide derivative, or both.

Adenine Nucleotides↗
PubMed1982

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments.

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.

Amino Acid Sequence↗
PubMed1979

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).

Adenosine Diphosphate Sugars↗
PubMed1985

Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments.

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.

Animals↗
PubMed1980

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Cyanogen bromide peptides.

Twelve cyanogen bromide peptides were isolated from S-carboxymethylated mitochondrial aspartate aminotransferase and their amino acid sequences were determined. These peptides were purified first by gel filtration on a Sephadex G-75 column, and then by gel filtration on Bio-Gel, or by ion exchange chromatography on a phosphocellulose column in the presence of 8 M urea, or by both methods. Small peptides were purified by paper chromatography. The cyanogen bromide peptides accounted for 367 of the 401 amino acid residues in the subunit of the enzyme. No peptide accounting for the other 34 residues was obtained in a homogeneous state, but peptide mixtures containing this particular peptide were analyzed by various procedures including Edman degradation and digestion with Staphylococcus aureus protease. The results accounted for all 401 amino acid residues.

Amino Acid Sequence↗
PubMed1985

[Primary structure of the OSCP protein that confers sensitivity to oligomycin on the mitochondrial H+-ATPase complex. I. Tryptic and cyanogen bromide peptides].

Trypsin and cyanogen bromide were used for cleavage of the OSCP preparations. The peptide mixtures thus formed were separated into individual components by a combination of various chromatographic procedures: gel filtration, ion exchange and paper chromatography, as well as reversed-phase HPLC. As a result, 31 tryptic peptides and 9 out of 10 possible cyanogen bromide peptides were isolated. Determination of the amino acid sequences of these peptide allowed the alignment of cyanogen bromide fragments in the polypeptide chain that shed light on the "architecture" of the protein molecule as a whole. It also afforded the overlappings for tryptic peptides, 16 in the N-terminal and 8 in the C-terminal portions of the molecule.

Adenosine Triphosphatases↗
PubMed1985

Template-directed synthesis of oligoadenylates using cyanogen bromide and diiminosuccinonitrile.

Cyanogen bromide was found to be a highly effective reagent for the coupling of oligoadenylates on a polyuridylic acid template in aqueous solution. Imidazole and divalent metal ions were required for the reaction. Hexaadenylic acid was converted to (pA)12, (pA)18, (pA)24, (pA)30 and (pA)36 in the presence of 0.01 M Co2+ and imidazole. When the reaction was carried out at 25 degrees C for 3 days, the oligomers were obtained in a 34% overall yield.

Adenine Nucleotides↗
PubMed1985

Destruction of cyanogen bromide and inorganic cyanides.

Cyanogen bromide in water and seven organic solvents and sodium cyanide in water may safely and efficiently (greater than 99.7%) be destroyed using sodium hydroxide (1 M) solution and commercially available sodium or calcium hypochlorite. Details are given of an analytical procedure which can be used to check the final reaction mixture for the presence of residual cyanogen bromide or cyanide.

Calcium↗
PubMed1977

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides.

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).

Amino Acid Sequence↗
PubMed1980

Complete amino acid sequence of human phosphoglycerate kinase. Cyanogen bromide peptides and complete amino acid sequence.

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.

Amino Acid Sequence↗
PubMed1975

On the primary structure of human plasminogen and plasmin. Purification and characterization of cyanogen-bromide fragments.

Most of the cyanogen bromide fragments obtained from human plasminogen and plasmin have been purified using combinations of gel filtration and ion-exchange chromatography. The purified fragments have been characterized by molecular weight determination (dodecyl sulphate electrophoresis), amino acid analysis, carbohydrate analysis and direct NH2-terminal amino acid sequence determination. Since some of the purified fragments were compounds with uncompletely cleaved methionyl bonds it was possible to clarify the organization of most of the cyanogen bromide fragments in the plasminogen molecule. The fragment containing the arginyl-valyl bond cleaved during the second step of the activation process is further identified. It is also shown that the microheterogeneity that normally exists in human plasminogen probably has its origin in several sites. One such site is situated in the light (B) chain of plasmin, while another is situated in the carboxyterminal part of the heavy (A) chain. Neither of these sites seems to contain sialic acid.

Amino Acid Sequence↗
PubMed1976

Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin.

Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.

Amino Acid Sequence↗
PubMed1985

Quantification and specific detection of collagenous proteins using an enzyme-linked immunosorbent assay and an immunoblotting for cyanogen bromide peptides.

A method for the detection of collagenous proteins within cyanogen bromide digests of tissues has been devised. The peptides produced by digestion with cyanogen bromide were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. They were stained on the filter by incubation first with antibodies to collagen and then with a second antibody covalently linked to horseradish peroxidase, 4-chloro-1-naphthol was added, and the bound enzyme was assayed. This procedure is useful for the identification and characterization of collagens of types I, III, IV, and V in tissues. In addition, we have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) which is convenient for quantifying collagens (types I, III, and IV) in tissues. In this kind of assay, soluble cyanogen bromide peptides compete with cyanogen bromide peptides adsorbed onto a solid-phase support for rabbit anti-collagen antibodies. We determined the amount of bound antibody by using goat anti-rabbit immunoglobulin G covalently conjugated to horseradish peroxidase and then provided a substrate for the enzymatic reaction. The sensitivity range of the ELISA is 0.09 micrograms/ml in the region of 90 to 10% binding.

Animals↗
PubMed1975

Structural studies on rabbit skeletal muscle actin. Ordering of the peptides produced by cleavage with cyanogen bromide.

The 17 peptides produced by cleavage of actin with cyanogen bromide have been ordered with regard to their sequence in the actin molecule. Tryptic digestion of actin followed by isolation of the methionine-containing "overlap" peptides permitted the unique alignment of most, but not all of the cyanogen bromide peptides. However, maleylation of the actin molecule followed by tryptic digestion and isolation of methionine-containing peptides from maleylated actin permitted the proper placement of the remaining cyanogen bromide peptides. The ordering of cyanogen bromide peptides, together with the amino acid sequence of the individual peptides, constitutes the entire amino acid sequence of rabbit skeletal muscle actin (ELZINGA, M., COLLINS, J. H., KUEHL, W. M., and ADENLSTEIN, R. S. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,2687-2691).

Actins↗