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In this paper, the boron‐modified polyferrocenylcarbosilanes (namely HBPFCS‐Bs) with hyperbranched structure, as single‐source‐precursors, were successfully prepared by using 9‐borabicyclo [3, 3, 1] nonane (9‐BBN) and polyferrocenylcarbosilanes (HBPFCSs) as starting materials. The obtained HBPFCS‐Bs were characterized using Fourier transform infrared spectroscopy (FT‐IR), nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC), providing that the boron element can be introduced into the HBPFCS chains by the hydroboration (B‐H/C = C) reaction. Then, the structural evolution of HBPFCS‐Bs and the boron‐containing SiFeC ceramics at different temperatures (1100–1500°C) was further investigated by thermogravimetric analysis, X‐ray diffraction, energy dispersive spectroscopy, scanning electron microscope, and transmission electron microscopy. The results show that the incorporation of a small amount of boron (1 wt%) into precursors significantly improves the properties of the final ceramics, including the significant increase of the ceramic yield, enhanced high‐temperature resistance, as well as the higher ceramic densification. Finally, the saturation magnetization of the HBPFCS‐Bs‐derived ceramics was also enhanced by the introduction of boron. [ABSTRACT FROM AUTHOR]

Five major sialyloligosaccharides and a sialylglycopeptide have been isolated from normal human urine by charcoal adsorption, gel filtration, ion-exchange chromatography, and paper chromatography. Structural studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, glycosidase treatments, and CrO3 oxidation indicated the Following structures for the compounds: 1, NcuAc(α2- 6)Gal(β1–4)GIe: 2. NeuAc(α2–6)Gal(β1–4)GIcNAc; 3. NeuAc(α2–3)Gal(β1–4)GIc; 4. NeuAc(α2–3)Gal(β1–4) GIcNAc: 5. NeuAc(α2–3)Gal(β1–3) [NeuAc(α2–6)]GalNAc; and 6. NeuAc(α2–3)Gal(β1–3) [NeuAc(α2–6)]GallNAc (α1-O)Ser. Compounds 4, 5, and 6 have not been described in a free form before. The presence of compound 5 in urine may suggest that it derives from glycoproteins through a catabolic pathway involving cleavage of the carbohydrate-peptide linkage by an endo-N-acetylgalactosaminidase. The predominating sialyloligosaccharides in urine were compounds 3 and 4. The predominance of the compounds with the sialyl(α2–3) linkage is of interest in view of the recent discovery of uropathogenic Escherchia coli strains with binding specificity for sialyl(α2–3)galactosides. [ABSTRACT FROM AUTHOR]

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity. Synopsis: The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control. The presented workflow is based on the concept of complex‐centric proteome profiling and combines size exclusion chromatography (SEC) with SWATH/DIA mass spectrometry.The implementation of the complex‐centric data analysis is supported by the computational framework CCprofiler.Application of the SEC‐SWATH‐MS workflow to HEK293 cells led the detection and quantification of subunit distribution of 462 distinct protein complexes containing 2,127 proteins and identified novel complex variants such as assembly intermediates.The interactive platform, SECexplorer is presented to support custom complex‐centric exploration of SEC‐SWATH‐MS datasets. The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control. [ABSTRACT FROM AUTHOR]

The N-linked oligosacchafides of C-reactive protein (CRP) from the arachnid Limulus polyphemus, the horseshoe crab, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High-voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P4 yielded five tractions. The oligosaccharide fractions were further fractionated using high-voltage borate paper electrophoresis and Dionex BioLC ion-exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man5GlcNAc2 (B-1), Man4GlcNAc2 (C-3) and one was the monoantennary structure Man3GlcNAc2 (D-1). The biantennary oligomannose structures B-1 and C-3 contained the structural unit Manα6Manα6R. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reposed in mammals, plants and the parasitic protozoa. [ABSTRACT FROM AUTHOR]

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojidmycin and A'-methyl-1-deoxynojidmycin [Hughes, R, C, Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544], Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [³H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean α-mannosidase. The data show that Glc3Man8GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2, Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosacchaddes to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases. [ABSTRACT FROM AUTHOR]

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9–204, 56–66, 71–77, 121–193, 126–177, 134–149, 145–158 and 159–164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a heroin 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 44000. 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. [ABSTRACT FROM AUTHOR]

The composition of lipopolysaccharide and polysaccharides isolated after acidic or alkaline degradation of lipopolysaccharide of Salmonella toucra has been investigated. The following analytical methods were used in this study: gel-filtration and ion-exchange techniques, paper and gas-liquid chromatography as well as spectrophotometric analysis. The products of the lipopolysaccharide degradation were fractionated on the Sephadex G-25 and Sephadex G-50 columns. Lipopolysaccharide and products of its degradation besides glucosamine, galactose, glucose, heptose(s) and 3-deoxy-D-manno-octulosonate described as ‘basal’ sugars, also contained N-acetylneuraminic acid and an unidentified amino sugar. [ABSTRACT FROM AUTHOR]

The interaction of actin with various cations has been studied at pH 7.6, at 20 °C, by means of gel filtration and centrifugation techniques. In addition to the single high-affinity site, five low-affinity sites with apparent association constants of 5-6 x 10³ M-1 for Ca2+, Mn2+ and Sr2+ and of 1.6 x 104 M-1 for Ni2+ have been found in the actin molecule. Unlike the other cations, Zn2+ appears to have three high-affinity sites from which it is not removed by Dowex 50 treatment, and eight low-affinity sites with an apparent association constant of 1.3 x 104 M -1. Competition experiments suggest that Ca2+, Sr2+, Mn2+ and Ni2+ are bound to the same low-affinity sites, some of which are also available for Zn2+. Zn2+ also binds to additional sites which are not available for the other cations. Binding of 4 mol of either Ca2+, Mn2+, Sr2+, Ni2+ or Zn2+ per mol of actin monomer at the low-affinity sites characterized above causes a complete transformation of the monomeric actin into polymers. The nonidentity of some of the 2+-binding sites with those for the other cations seems to explain structural differences between the polymers formed in the presence of this cation and those produced upon binding of Mn2+, Ni2+ or alkaline earth metals, described in the preceding paper in this journal. The results suggest that formation of the double-stranded F-actin filaments requires the neutralization of negative charge at specific sites in the monomer molecules. The aggregation of F-actin filaments into paracrystals observed at pH 7.6 in the presence of Ca2+, Mg2+, Sr2+ or Mn2+ at millimolar concentrations seems to involve the binding of these cations to a third class of binding sites with apparent association constants of the order of 10² M-1. The binding parameters to this class of sites could not be determined by the methods used in this work. [ABSTRACT FROM AUTHOR]

1. From 90 1 erythrocytes 200 mg glutathione reductase was purified in four steps with an overall yield of 40%. The specific activity rose from 3.71 U glutathione reductase per g hemoglobin in the erythrocytes to 235 U/mg protein in the isolated enzyme. The purification procedure which was completed in 5 days, included (a) denaturation of hemoglobin by a mixture of butan-1-oI and chloroform [Scott. E. M. (1976) Prep. Biochem. 6, 147-152]. (b) precipitation of glutathione reductase with acetone, (c) extraction of the precipitate and fractionation of the extract with ammonium sulfate, (d) affinity chromatography on adenosine-2',5'-bisphosphate -Sepharose 4B. 2. The 280/460 nm absorbance ratio was found to be 5.9 for the purified enzyme; all other studies on physical and chemical properties of the enzyme confirmed the results of Worthington and Rosemeyer [Eur. J. Biochem. 67, 231-238 (1976) and earlier papers]. 3. The new purification procedure provided crystals suitable for X-ray diffraction analysis at high resolution. Furthermore sufficient amounts for sequencing the enzyme were isolated. As a first step in the determination of the protein's primary structure, CNBr-produced fragments of the carboxymethyiated enzyme were fractionated. 4. A chain segment containing the catalytic disulfide of the native protein was isolated. Its sequence was determined to be Leu-Gly-Gly-Thr-Cys-Val-Asn-Val-Gly-Cys-Val-Pro-Lys. An identical sequence is present in yeast glutathione reductase, and lipoamide dehydrogenases contain an active site peptide with very similar sequence [Williams, C. H. Jr (1976) The Enzymes 13, 89 - 173]. Thus the long-recognized mechanistic similarities between glutathione reductases and lipoamide dehydrogenases are reinforced by sequence similarities around the two redox-active cysteine residues. The sequenced regions are, however, too short to allow speculations on phylogenetic relationships within this category of proteins. [ABSTRACT FROM AUTHOR]

Three toxins (CM-8, CM-11, and CM-13 a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose, Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 6t residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-tt resemble the corresponding properties of the cytotoxin group, Tile sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group, [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(α2-6)Gal(β1-4)GlcNAc(α)-p; NeuAc(α2-3)Gal(β1-4)GlcNAc(α)-P; NeuAc(α2-3)Gal(β1-3)GalNAc(α)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates. [ABSTRACT FROM AUTHOR]

Sialyl-glycopeptides containing an O-glycosidically linked tetrasaccharide chain were obtained from the urine of a patient suffering from mucolipidosis I. Isolation of these compounds was achieved by gel filtration, ion-exchange chromatography and preparative paper chromatography. Their structures were determined by a combination of carbohydrate and amino acid analysis, dansylation, periodate oxidation, methylation studies, enzymatic hydrolysis and ¹H-NMR spectroscopy, to be as follows: ... wherein R = peptide linked through -Thr-, Ser-, -Ser-Thr- or -Thr-Ser-. The finding of these glycopeptides in urine shows that mucolipidosis I is characterized by a general "glycoprotein-specific" sialidase deficiency. The possibility of the existence of a human endo-α-N-acetylgalactosaminidase is discussed. [ABSTRACT FROM AUTHOR]

In a recent paper in this journal it was shown that human ceruloplasmin contains a single continuous polypeptide chain composed of about 1050 amino acid residues. Together with the carbohydrate this accounts for the molecular weight of the native protein 132000. In contrast to this protein ceruloplasmin has been reported to contain two subunits of molecular weight 70000-80000. This was the reason for performing a direct comparison of polypeptide-chains from ceruloplasmin from difference mammalian sera. Ceruloplasmin was prepared from sera of man, pig, horse and rabbit. The reduced and alkylated proteins were gel filtered on a calibrated column of 6% agarose in 6M guanidine hydrochloride. The four proteins all contained a single main component cluting in the same position within errors of measurements. It is concluded that the four proteins investigated all contain a single polypeptide chain of very similar size. [ABSTRACT FROM AUTHOR]

α-Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH-4.0 precipitation, followed by gel filtration with Sephadex G-100. A final purification stage using DEAE-cellulose was necessary in some preparations. Peptides derived from the reduced, S-aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypain, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high-voltage paper electrophoresis. From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α-laotaIbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α-Iactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed. [ABSTRACT FROM AUTHOR]

An immunoglobulin M from Waldenström's disease (IgM Ga) was hydrolysed by papain and a glycopeptide fraction was isolated by gel filtration on Sephadex G-100. This fraction was then successively resolved into fourteen glycopeptides by gel filtration on Sephadex G-50 and by preparative paper electrophoresis at pH 1.9. The amino acid and carbohydrate content of these glycopeptides was determined by ionexchange chromatography in an autoanalyzer. All of them contain aspartic acid and serine, but they are quite different from one another, in amino acid composition and sequence about, the carbohydrate-peptide linkage, especially with respect to histidine, tyrosine, threonine, valine, glutamic acid, lysine and proline. Furthermore, a number of glycopeptides contain only mannose and glucosamine, while others contain in addition, galactose, fucose, and N-acetylneuraminic acid, in variable amounts. These results suggest the presence of several linkage sites of the oligosaccharide chains on the H-chain and the true heterogeneity of these oligosaccharide chains. The peptide sequence of a few glyeopeptides was reported, especially the following: r-Asn(carbohydrates)-Ser-Thr, also present, in the Fc fragment of Immunoglobulin G. [ABSTRACT FROM AUTHOR]

‘Natural’ opsonins to Salmonella typhimurium in serum from foetal and natal rats have been shown to be in part macroglobulins. The titre of these opsonins was shown to increase in the young natal animals. [ABSTRACT FROM AUTHOR]