High-voltage Paper Electrophoresis Research Articles

The isolation and identification of 3,4-dihydroxybenzoic acid formed by nitrogen-fixingAzomonas macrocytogenes

The mechanism of iron acquisition was studied in nitrogen-fixing Azomonas macrocytogenes. Spent solid agar plating medium samples from Fe-deficient and Fe-sufficient cultures were subjected to the high voltage paper electrophoresis siderophore assay. A yellow-green fluorescent peptide was elicited only under conditions of iron deficiency, whereas a novel dark blue fluorescent, Arnowpositive phenolic compound was detected under both Fe-deficient and Fe-sufficient conditions. Both compounds formed colored complexes with ferric iron. SDS-PAGE analysis of outer envelope protein preparations revealed the hyperproduction of an 83 kDa protein under iron-limiting conditions. Chemical analysis indicated that the phenolate was 3,4-dihydroxybenzoic acid (protocatechuic acid), a compound previously unreported as an extracellular product of a diazotroph.

The isolation and identification of 3,4-dihydroxybenzoic acid formed by nitrogen-fixing Azomonas macrocytogenes

The mechanism of iron acquisition was studied in nitrogen-fixing Azomonas macrocytogenes. Spent solid agar plating medium samples from Fe-deficient and Fe-sufficient cultures were subjected to the high voltage paper electrophoresis siderophore assay. A yellow-green fluorescent peptide was elicited only under conditions of iron deficiency, whereas a novel dark blue fluorescent, Arnowpositive phenolic compound was detected under both Fe-deficient and Fe-sufficient conditions. Both compounds formed colored complexes with ferric iron. SDS-PAGE analysis of outer envelope protein preparations revealed the hyperproduction of an 83 kDa protein under iron-limiting conditions. Chemical analysis indicated that the phenolate was 3,4-dihydroxybenzoic acid (protocatechuic acid), a compound previously unreported as an extracellular product of a diazotroph.

Reaction products formed after strong acid treatment of α-amino-δ-hydroxyvaleric acid

High-voltage paper electrophoresis and analytical and preparative amino acid ion-exchange chromatography were used to investigate the products resulting from strong acid treatment and strong acid treatment and subsequent alkalinization of α-amino-δ-hydroxyvaleric acid. After strong acid treatment, α-amino-δ-hydroxyvaleric acid and proline, as well as several unidentified components, presumably lactones, were recovered. No definitive identification of the putative unstable lactones was accomplished either by physical-chemical means or by ion-exchange chromatography or high-voltage paper electrophoresis. Nevertheless, after acid treatment and alkalinization, only α-amino-δ-hydroxyvaleric acid and proline were recovered, in approximately equal amounts. The formation of such large amounts of proline from the acid treatment and alkalinization of α-amino-δ-hydroxyvaleric acid is therefore an important consideration in quantitating the number of γ-glutamyl phosphate residues present in various proteins.

99mTc-1,7-bis (2-pyridyl)-2,6-diazaheptane (DPTN) complexes: Production yield and stability in slightly basic media

The reduction of99mTc by SnCl2 in the presence of 1,7-bis (2-pyridyl)-2,6-diazaheptane (DPTN) has been investigated in borate buffer solutions with pHs ranging from 6.7 to 9.5. The resulting cationic, neutral and anionic complexes, in addition to TcO2 and TcO 4 − impurities, were separated by high voltage paper electrophoresis. The cationic complex turned into neutral and then into an anionic form via hydrolysis. These three species were also separated by gel filtration on a Bio-Gel P-2 column; their formation yields, stability, and the rate of their hydrolysis are presented as a function of the pH, and the results are discussed.

UDP-GalNAc : GalNAc-mucin α- N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used as an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin α-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAcα→ GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis. GalNAc transferase Human intestinal cancerous tissue Bovine submaxillary gland mucin O- Glycosidically linked sugar chain

Direct chemical evidence for the mixed anhydride intermediate of carboxypeptidase A in ester and peptide hydrolysis

Carboxypeptidase A was incubated at −60°C with an excess of O -( trans - p -chlorocinnamoyl)-L-phenyllactate, O -(hippuryl)-glycolate or N -(hippuryl)-L-phenylalanine. After rapid denaturation with trichloracetic acid the precipitated protein was reduced with [ 3H]NaCNBH 3. 3H Labeled enzyme was isolated by gel chromatography on Sephadex G-25. After complete acid hydrolysis the specific label within the protein was identified by high voltage paper electrophoresis and paper chromatography as [ 3H]2-amino-5-hydroxyvaleric acid, the reduction product of a γ-acylated glutamic acid. These results give strong evidence that a mixed anhydride intermediate is formed, which for the first time was identified during the hydrolysis of classical ester as well as peptide substrates by direct chemical means.

The structure of a fucose-containingO-glycosidic carbohydrate chain of human platelet glycocalicin

The hydrazinolysis procedure currently used for the release ofN-glycosidic carbohydrate chains was applied to glycocalicin. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis into a neutral (5%) and several acidic fractions. The neutral compounds were passed over Bio-Gel P-4. SomeN-glycosidic oligosaccharide-alditols, of theN-acetyllactosamine type as well as of the oligomannoside type, were found to be present. However, oligosaccharide-alditols derived fromO-glycosidic carbohydrate chains were also found, indicating a partial cleavage of GalNAc1-OSer/Thr linkages under the hydrazinolysis conditions applied. One of the neutralO-glycosidic components was characterized, by 500-MHz1H-NMR spectroscopy in combination with sugar analysis, as the following pentasaccharidealditol: In addition the afuco analogue of this compound was obtained.

Structural relatedness of three ion-transport adenosine triphosphatases around their active sites of phosphorylation.

Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation. The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase. A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated. For most of the experiments, the preparations were phosphorylated from [gamma-32P]ATP, denatured in acid, and subjected to proteolytic digestion. Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents. In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with [3H]borohydride. A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase. This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum. The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP. The corresponding sequence was different from that of the three ATPases. An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R. N. A. H., and McElhaney, R. N. (1983) Biochim. Biophys. Acta 735, 113-122).

Characterization of 2-substituted pyridinium salts by high-voltage paper electrophoresis

Characterization of 2-substituted pyridinium salts by high-voltage paper electrophoresis

Evidence of tyrosine kinase activity in the photosynthetic bacterium Rhodospirillum rubrum

The photosynthetic bacterium Rohodospirillum rubrum evidence tyrosine protein phosphorylation under photoautotrophic conditions in the presence of { 32 P}Pi. The stability to alkaline treatment of the { 32 P} bound to the cell-free extract proteins suggested that tyrosine residues were carrying the labelling. One- and two-dimensional high voltage paper electrophoresis analysis revealed that such extracts do contain { 32 P}-phosphotyrosine residues. Furthermore, the association of alkali stable { 32 P} bound to specific proteins of the cell-free extract was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with KOH treatment of the gel. A definite argument in favor of protein kinase(s) phosphorylating tyrosine residues in R.rubrum proteins was obtained by partial purification of a tyrosine kinase activity from cell-free extract capable of phosphorylating synthetic peptides that only contain a single tyrosine residue as phosphate acceptor.

Methyl ethyl ketone extraction of Tc species

Methyl ethyl ketone extraction of technetium species from aqueous solutions of neutron irradiated ammonium molybdate crystals was studied; the two species extracted were separated by high voltage paper electrophoresis. The one was the99mTcO 4 − ion and the other, a99mTc non-charged immobile species, probably TcO2.aq, which concentrated at the point of application on the electrophoresis paper strip.

The structure of the polysaccharide moiety of Rhodopseudomonas sphaeroides ATCC 17023 lipopolysaccharide.

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.

Extraction, purification and identification of cytidine 3',5'-cyclic monophosphate from rat tissues.

The large-scale extraction and purification to homogeneity of cyclic CMP and its unequivocal identification are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and boronate column chromatography, polyacrylamide-gel column electrophoresis and high-voltage paper electrophoresis. The purified sample co-chromatographed with authentic cyclic CMP on t.l.c. and high-pressure liquid chromatography and was positive in a cyclic CMP radio-immunoassay. The u.v., i.r. and p.m.r. spectra were each essentially identical with those of authentic cyclic CMP. Fast-atom bombardment of authentic cyclic CMP yielded a mass spectrum containing a molecular protonated ion: mass-ion-kinetic-energy scanning of this ion produced a spectrum unique to 3',5'-cyclic CMP. The extracted nucleotide produced an identical mass-ion-kinetic-energy spectrum.

Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments.

The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.

Selenium speciation in the soil solution and its relevance to plant uptake

AbstractThe relationship between soil and plant selenium has been investigated using two 75SeO3 spiked silt loam soils from the Hoosfield series with long‐term equilibration pH values of 4.5 and 7. Lolium perenne plants were grown in the soils over a 15‐week period, and seven harvests were made. Plant samples and soil solutions were assayed for radioactivity, and selenium compounds found in the soil solutions separated by high voltage paper electrophoresis. Throughout the 15‐week experimental period selenium speciation was observed to change in soil solution. Samples collected at day 2 from pH 4.5 soil revealed that selenate accounted for 71% and selenite 8% of the soluble selenium compounds present, whereas for the pH 7 soil the relative percentages were 51 and 23% respectively. After 105 days selenate accounted for 22% and selenite 20% at pH 4.5, and 12 and 22% respectively at pH 7. The occurrence of selenoglutathione was noted in the soil solution and its concentration increased significantly with time. Statistically significant correlations (r=0.82) were found between the concentration of selenium in Lolium perenne at harvest and selenate selenium removed from the soil solution.

Primary structure of the major glycans of the N-acetyllactosamine type derived from the human immunoglobulins M from two patients with Waldenström's macroglobulinemia

The carbohydrate chains of the pathological human immunoglobulins M from two patients with Waldenström's macroglobulinemia were released by hydrazinolysis. The N-acetyllactosamine-type glycans were obtained by affinity chromatography on concanavalin A and fractionated by high-voltage paper electrophoresis. The primary structure of the major compounds was elucidated on the basis of carbohydrate analysis, methylation analysis, including mass-spectrometry, and 500 MHz 1H-NMR spectroscopy. For both patients, this appeared to be a monosialyl monofucosyl biantennary structure; the compounds differed by the presence of an intersecting N-acetylglucosamine residue.

Branch specificity of purified rat liver Golgi UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase. Preferential transfer of of galactose on the GlcNAc beta 1,2-Man alpha 1,3-branch of a complex biantennary Asn-linked oligosaccharide.

In the final stages of the terminal glycosylation of N-linked complex oligosaccharides, UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase (galactosyltransferase) transfers galactose (Gal) onto the N-acetylglucosamine (GlcNAc) residue of each branch of a biantennary oligosaccharide. Purified rat liver Golgi galactosyltransferase was used with GlcNAc beta 1,2-Man alpha 1,6-(GlcNAc beta 1,2-Man alpha 1,3-)-Man beta 1,4-GlcNAc beta 1,4-(Fuc alpha 1,6-)-GlcNAc-Asn in order to determine the sequence of addition of Gal residues to the biantennary oligosaccharide. The different galactosylated products were separated by concanavalin A affinity chromatography and high voltage paper electrophoresis in borate. It was found that Gal was transferred at a much faster rate to the GlcNAc beta 1,2-Man alpha 1,3-branch than to the GlcNAc beta 1,2-Man alpha 1,6-branch, i.e. k1 was at least 5 times larger than k2. Also, k3 was larger than k4, indicating that most of the digalactosylated product "GG" was formed by the sequential addition of Gal to the Man alpha 1,3-branch followed by addition to the Man alpha 1,6-branch. The preferential galactosylation of the GlcNAc beta 1,2-Man alpha 1,3-branch may explain the formation of the asymmetrical oligosaccharides found in bovine and human IgG.

Arabinogalactan-proteins reacting with eel anti-H agglutinin from leaves of cruciferous plants.

Glycoconjugates reacting with eel anti-H agglutinin were purified from extracts of leaves of three species of cruciferous plants (radish, turnip, and rape) by precipitation with ethanol, ionexchange chromatography, and gel filtration. High voltage paper electrophoresis or ultracentrifugal analysis revealed that the purified specimens were homogeneous.Their apparent molecular weights were estimated to range from 0.5 to 1.5×105. They consisted of a novel L-fucose-containing acidic arabinogalactan-protein composed of residues of L-arabinose, D-galactose, L-fucose, 4-O-methyl-Dglucuronic acid, and D-glucuronic acid in similar molar proportions, and containing polypeptide portions with abundant hydroxyproline, serine, threonine, and alanine. All the arabinogalactanproteins exhibited potent inhibitory activity against the hemagglutination of human O erythrocytes by eel anti-H agglutinin.

Effects of various strengths of acids on the electrophoretic behavior of weak bases in a non-aqueous solvent

By the addition of an appropriate strength and concentration of acid which is in acetonitrile—sulfolane (9:1) containing a small amount of tetraethylammonium perchlorate as background electrolyte, weak organic nitrogen bases showed differential migration in high-voltage paper electrophoresis. Dichloro- and trichloro-acetic acids were effective for separating 5,6-benzoquinoline, 7,8-benzoquinoline and 1,10-phenanthroline. The use of potentiometric and spectrophotometric titration data to predict the electrophoretic behavior was very limited due to adsorption of the free bases by paper.

Reductive Isopropylation of Amino Groups in Lysine Containing Peptides

Abstract Model peptides, Gly-Gly-Lys-Arg, Arg-Lys-Asp-Val-Tyr, and Pro-Gly-Lys-Ala-Arg were reductively alkylated with [2H6]acetone and sodium borohydride to assess the effects on peptide behavior. Lysine residues were converted to ∊-N-isopropyllysine which eluted between phenylalanine and histidine on amino acid analysis. Amino terminal groups were also modified to an extent which depended on the particular peptide (glycine 100%, arginine 30%, and proline 10%-20%). High voltage paper electrophoresis of native and isopropylated peptides showed similar properties except for minor decreases in the mobility of the modified peptides due mainly to increased molecular weight. Isopropyllysine was not an effective substrate for trypsin, and α-N-isopropyl-amino acids did not form dansyl chloride derivatives. These findings should aid in the location, by peptide mapping techniques, of specific modified residues in reductively isopropylated proteins.