Showing papers on "Paper chromatography published in 1987"

Major fragment of soluble peptidoglycan released from growing Bordetella pertussis is tracheal cytotoxin

Abstract: Bordetella pertussis is known to release a factor which promotes the loss of ciliated respiratory epithelium and copurifies with a soluble peptidoglycan (PG) fragment termed tracheal cytotoxin (TCT). The objective of this study was to determine whether pertussis organisms turn over and release PG derivatives in addition to TCT. B. pertussis Tohama (phase III) was grown in liquid Stainer-Scholte medium containing [3H]diaminopimelic acid (DAP) to label PG specifically, washed to remove free label, and suspended in fresh medium without [3H]DAP. Molecular sieve chromatography of supernatants obtained from such cultures revealed a single included peak of 3H, the elution volume of which corresponded roughly to a disaccharide peptide monomer standard (ca. 10(3) daltons). This material (i) contained [3H]DAP in acid-hydrolyzable linkage, (ii) comigrated with 1,6-anhydro-N-acetylmuramic acid-containing disaccharide peptides on paper chromatography, (iii) was resistant to degradation by mild alkali, and (iv) was indistinguishable from authentic TCT by high-voltage paper electrophoresis and two reversed-phase high-performance liquid chromatography systems. Together, the data suggest that B. pertussis releases a markedly homogeneous set of PG fragments, consisting principally of TCT, and that TCT is possibly a nonreducing, anhydromuramic acid-containing fragment or a cyclic PG derivative.

Cytokinins in cut carnation flowers: I. The complex in ovaries

Abstract: Tentative identification of the cytokinins present in extracts of Dianthus caryophyllus ovaries using High Performance Liquid Chromatography and Radioimmunoassay techniques, revealed the presence of trans-ribosylzeatin, trans-zeatin, dihydrozeatin and N6 (△2-isopentenyl)adenine. In addition slow moving compounds (paper chromatography) which could be hydrolysed by β-glucosidase were also detected. After hydrolysis the active compounds co-chromatographed with zeatin and ribosylzeatin.

Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid.

Abstract: A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus The enzyme catalyzes the transfer of methyl groups from [14C]S-adenosylmethionine to HAA, but does not catalyze the methylation of 3-hydroxy-DL-kynurenine Enzyme, substrate, time, and pH dependencies for the methyl transfer reaction were examined Reaction products obtained from scaled-up reaction mixtures were fractionated by chromatography on Dowex 1, and the Dowex 1 fractions were examined by paper and thin-layer chromatography One Dowex fraction was shown to contain a radioactive product with the chromatographic properties of 4-methyl-3-hydroxyanthranilic acid (MHA), a known intermediate in the biosynthesis of actinomycin Available evidence indicates that the conversion of HAA to MHA is an early step in the biosynthesis of actinomycin by S antibioticus and other actinomycin-producing streptomycetes

Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

Abstract: Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.

Conversion of N,N-dimethylaniline to N,N-dimethylaniline-N-oxide by a cytosolic flavin-containing enzyme from Trypanosoma cruzi.

Abstract: A cytosolic NADPH-dependent FAD-containing enzyme purified from Trypanosoma cruzi epimastigotes [Kuwahara, White, and Agosin: Biochem. Biophys. Res. Commun. 124, 121 (1984); Arch. Biochem. Biophys. 239, 18 (1985)] catalyzes the conversion of N,N-dimethylaniline to its corresponding N-oxide. The identity of the product has been established by high performance liquid chromatography and paper chromatography elution patterns and by electron impact spectrometry. The oxidation of N,N-dimethylaniline was determined by following the oxidation of NADPH spectrophotometrically, and a double reciprocal plot of reaction velocity vs. substrate concentration was prepared. At an optimum pH of 8.0, the plot resulted in Km and Vmax values of 56 microM and 114 nmol X min-1 X mg of protein-1, respectively. The oxidative activity of the enzyme suggests that it may be involved in detoxication processes which may contribute to the resistance of T. cruzi to known antiprotozoal drugs.

Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells.

Abstract: Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.

Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

Abstract: Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

Thin Layer Chromatography

Abstract: Introduction: Historical Development from Paper Chromatography, General Experimental Arrangements Stationary Phase: Adsorbants, High Performance Thin Layer Chromatography Practical Techniques: Spotting of Plates, Preparation of Solvent Tanks, Methods of Plate Development, Visualisation Applications: Qualitative TLC, Quantitative TLC, Preparative TLC, Advantages and Disadvantages of TLC

Separation of unsaturated chondroitin sulfate disaccharides in thin-layer chromatography on silica gel and their quantitative determination by densitometry.

Abstract: A simple and rapid method has been developed to determine the relative amounts of 4-and 6-sulfate in chondroitin sulfate or proteoglycan preparations. Unsaturated disaccharide products derived from the samples by chondroitinase ABC digestion in 0.01 M Tri-HCl buffer (pH 8.0) were applied to a silica gel-coated thin-layer chromatographic plate and separated in a solvent system of n-propanol-isopropanol-n-butanol-water (55 : 20 : 5 : 20, v/v) containing 0.04M NaCl and 0.01 M ammonia without a desalting step, which was necessary in paper chromatography or thin-layer chromatography on cellulose. The developing time was as short as 2-2.5 h at room temperature. After staining of the separated products with a carbazole reagent followed by immersing the plate in liquid paraffin, the ratio of monosulfated disaccharides, generally found as major components in chondroitin sulfate chains, was estimated by densitometry.

Glyceraldehyde-3-phosphate dehydrogenase. Investigation on the regions responsible for self-assembly of subunits.

Abstract: 1. In glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) the four S-loop form the core of the tetramer. 2. Amino acid sequence of the S-loop of the regions of GAPDH from carp muscle was established through the analysis of tryptic digests of the enzyme treated alternatively with bromocyanate and o-iodosobenzoic acid. 3. Enzyme had been oxidized with performic acid. After treatment with trypsin the peptide mixture was fractionated into fragments. 4. CNBr cleavage of this enzyme was performed after S-carboxymethylation. The respective cyanogen bromide fragments have been isolated and characterized. 5. The procedure of protein fragmentation by o-iodosobenzoic acid used to split tryptophanyl peptide bonds. 6. Each peptide obtained after enzymatic or chemical fragmentation was purified to homogeneity by Bio-Gel or Sephadex chromatography, high voltage electrophoresis and descending paper chromatography and characterized by electrochromatography, N- and C-terminal sequence and amino acid composition. 7. The results are compared with those obtained from studies on GAPDH from other sources.

Preparative Isolation of Purine Oligonucleotides from Partial Hydrolyzates of Depyrimidinated DNA

Abstract: DNA is chemically degraded to a mixture of pure oligonucleotides. From the resulting partial hydrolyzate, defined oligonucleotides with two to six monomer units and defined mixtures of sequence-isomeric purine oligonucleotides are isolated in preparative amounts. The partial hydrolyzate is fractionated by a chromatographic separation route which uses anion-exchange chromatography on DEAE- and QAE-Sephadex at different pH values, paper chromatography, and reversed-phase HPLC. The sequence of the isolated oligonucleotides and the composition of the mixtures of sequence-isomers were determined from chromatographic data, absorption characteristics, enzymatic degradation, and “fingerprints.”

Rapid Analysis of the Food Colors in Foodstuffs Using Polyamide

Abstract: A rapid and simple analysis procedure involving a batch system using polyamide (nylon-6 powder) was devised for the detection of food colors. The food colors were extracted from food with a mixture of 1% ammonia water and ethanol (4:6). The extract was filtered, and the filtrate was diluted two or three times with water. The colors in the solution were absorbed on a small amount of polyamide (10-30mg) in acid (pH 4-5). After filtration and rinsing with hot water, the polyamide was suspended and eluted with a small quantity (about 100μl) of a mixture of 28% ammonia water and ethanol (4:6). After five min, the supernatant was used for paper chromatography and thin layer chromatography on a silica gel plate or a polyamide sheet. The solvent systems for the chromatographies were as follows: paper chromatography, butanol-ethanol-1% ammonia water (6:2:3); silica gell thin layer chromatography, ethyl acetate-ethanol-28% ammonia water (3.3:1:1) or (4.5:1:0.7); polyamide thin layer chromatography, methanol-ethanol-isoamyl alcohol-28% ammonia water (15:10:5:3). The systems containing ammonia as described above were required for separation of the food colors. Compared with the wool dyeing method, the presented method did not require evaporation of alcohol, heating for adsorption or elution of the food colors or concentration of the eluate. It is rapid, simple and precise for the detection of the food colors in various colored foods.