Bio-Gel P2 Chromatography Research Articles

Structural Analysis of Gluco-Oligosaccharides Produced by Leuconostoc lactis and Their Prebiotic Effect.

Leuconostoc lactis CCK940, which exhibits glycosyltransferase activity, produces oligosaccharides using sucrose and maltose as donor and receptor molecules, respectively. The oligosaccharides produced were purified by Bio-gel P2 chromatography and the purified oligosaccharides (CCK-oligosaccharides) consisted of only glucose. 1H-NMR analysis revealed that the CCK-oligosaccharides were composed of 77.6% α-1,6 and 22.4% α-1,4 glycosidic linkages, and the molecular weight of the CCK-oligosaccharides was found to be 9.42 × 102 Da. To determine the prebiotic effect of the CCK-oligosaccharides, various carbon sources were added in modified media. Growth of six probiotic strains, Lactobacillus casei, L. pentosus, L. plantarum, Weissella cibaria, Bifidobacterim animalis, and Saccharomyces cerevisiae, was better when the CCK-oligosaccharides were used as the sole carbon source compared to fructo-oligosaccharides, which are widely used as prebiotics. These results showed that the CCK-oligosaccharides produced from Leu. lactis CCK940 could serve as good candidates for novel prebiotics.

The synergistic anti-inflammatory activities of agaro-oligosaccharides with different degrees of polymerization

The anti-inflammatory activities of agaro-oligosaccharide (AOS) fractions with different degrees of polymerization (DPs) were compared, and their anti-inflammatory mechanisms were investigated. Total AOS (F0) was prepared from the agar of Gracilaria lemaneiformis, and its three separated fractions of high DP (F1), medium DP (F2), and low DP (F3) were obtained using Bio-Gel P2 chromatography. F2 showed a high anti-inflammatory activity but also a potential cytotoxicity, while F1 and F3 showed no cytotoxicity but weak anti-inflammatory activities. The total AOS fractions of F0 exhibited a higher anti-inflammatory activity than the summarized activities of the separated fractions; moreover, the combination of the three fractions significantly alleviated the potential cytotoxicity of F2. Thus, the synergistic anti-inflammatory effects of the AOS fractions were revealed. Furthermore, the anti-inflammatory mechanisms of F0 were investigated both in lipopolysaccharide (LPS)-induced RAW 264.7 cells in vitro and LPS-induced zebrafish embryos in vivo. F0 inhibited NO production and PGE2 expression via the downregulation of iNOS and COX-2 expression, and it suppressed TNF-α, IL-1β, and IL-6 productions at the protein and mRNA levels in RAW 264.7 cells. It is the first time that the zebrafish model was used for the determination of anti-inflammatory activities of AOS, and F0 showed to suppress the productions of NO and ROS by exerting a protective effect against the phenotypic changes and the cell damage caused by LPS. This is the first report of the synergistic anti-inflammatory activities of AOS fractions. The result revealed that AOS without separation and purification showed a favorable anti-inflammatory activity, which was easy to be produced and had potential application in the food, pharmaceutical, and cosmetic industries.

Molecular cloning and expression of a new α-neoagarobiose hydrolase from Agarivorans gilvus WH0801 and enzymatic production of 3,6-anhydro-l-galactose.

A new α-neoagarobiose hydrolase (NABH) called AgaWH117 was cloned from Agarivorans gilvus WH0801. The gene encoding this hydrolase consists of 1,086bp and encodes a protein containing 361 amino acids. This new NABH showed 74% amino acid sequence identity with other known NABHs. The molecular mass of the recombinant AgaWH117 was estimated to be 41kDa. Purified AgaWH117 showed endolytic activity during neoagarobiose degradation, yielding 3,6-anhydro-l-galactose (l-AHG) and d-galactose as products. It showed a maximum activity at a temperature of 30°C and a pH of 6.0 and was stable at temperatures below 30°C. Its Km and Vmax values were 2.094mg/mL and 6.982U/mg, respectively. The cloning strategy used and AgaWH117 isolated in this study will provide information on the saccharification process of marine biomass. This study provides a method to produce l-AHG from agarose by using AgaWH117 without an acid and describes its one-step purification by using Bio-Gel P2 chromatography.

PURIFICATION AND CHARACTERIZATION OF A CELLULASE FROM BACILLUS SUBTILIS YJ1

Broth incubated with Bacillus subtilis YJ1 at 37°C for 36 h was collected and removed bacterial cells by passing through a 0.45 µm membrane. A cellulase was purified to electrophoretical homogeneity by ammonium sulfate precipitation, Macro-Prep ion exchange and Bio-Gel P-100 chromatography. The recovery and purification fold were 9.7% and 289, respectively, after Bio-Gel P-100 chromatograph. The purified cellulase, with a molecular mass (M) of 32.5 kDa, had an optimal pH and temperature at 6.0 and 50-60°C, respectively. It was stable at pH 6.0-7.5 and <50°C. The purified cellulase was activated by Mn 2+ and Co 2+ , but inhibited by SDS, p-CMB, DTT, Hg + , Cd 2+ , Fe 2+ and Fe 3+ . According to substrate specificity, the purified cellulase has high specificity to CMC and was considered to be an endo-1,4-glucanase.

Type I arabinogalactan contains β- d-Gal p-(1→3)-β- d-Gal p structural elements

Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a β- d-Gal p-(1→4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all 1H and 13C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a β-(1→3)-linkage instead of the expected β-(1→4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: β-Gal p-(1→4)-β-Gal p-(1→4)-β-Gal p-(1→4)-α/β-Gal p-OH and β-Gal p-(1→4)-β-Gal p-(1→4)-β-Gal p-(1→3)-α/β-Gal p-OH. The fact that the deviating β-(1→3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The β-(1→3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion).

Structure of polysaccharide from Polygonatum cyrtonema Hua and the antiherpetic activity of its hydrolyzed fragments

A neutral polysaccharide named PD was isolated from the traditional Chinese medicinal herb, Polygonatum cyrtonema Hua. Five fragments were isolated by Bio-Gel P4 chromatography from hydrolysates of PD. Using assays of cytopathic effect inhibition, neutral red dye uptake and plaque forming inhibition, it was proved that the fragments with degree of polymerization (DP) of 4 and 5 were the shortest ones which retained the activity against herpes simplex virus type 2 (HSV-2) in vero cell culture. The structures of PD and one of its activity-retaining fragments, B3, were determined by permethylation followed with reductive cleavage, mass spectrometry and nuclear magnetic resonance spectrometry. It was shown that PD was a branched fructan with average DP of 28. There was one two-residue side chain composed of (2 → 6)-linked β- d-fructofuranosyl (Fru f) residues every three (2 → 1)-linked β- d-Fru f residues in the backbone of PD, whereas B3 was a mixture containing 1-kestose and neokestose series of oligosaccharides of DP 3–5 without branches.

Evaluation of musk by enzyme-linked immunosorbent assay.

We report the development of enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of a unique musk protein (MP-1) in musk samples. Musk defatted with ethyl acetate/methanol (9:1, v/v) was dipped in cold water and ammonium sulfate was added to the supernatant up to 85% saturation. The resulting precipitate was applied to a Bio-Gel P-100 chromatography. The fraction eluted at the void region was collected and it was consecutively purified by affinity chromatography on a DEAE Affi-Gel Blue and on anion-exchange columns containing DEAE-Sepharose CL-6B. This protein was determined to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions with an apparent molecular weight of 35000 Da and was called as musk protein-1 (MP-1). Polyclonal antibodies of MP-1 were produced by injecting it into a rabbit. These antibodies were reactive to the aqueous extract of musk and the pure antigen. The ELISA could be applied to detect nano gram quantities of the antigen in musk samples. This method made it possible to distinguish musk samples from different origins.

Inhibition of endive (Cichorium endivia L.) polyphenoloxidase by a Carica papaya latex preparation

When endive polyphenoloxidase (PPO) was incubated with a crude papaya latex extract, it rapidly lost its activity. Inactivation was ascribed to thermostable nonenzymatic factors of low molecular weight. These factors were partially purified by a two step protocol including gel filtration chromatography on Biogel P2 and ion exchange chromatography using DEAE Sephadex A25. The PPO‐inactivation rate was first order, when either inactivating agent or proton concentration was evaluated. Inactivation could be partially reversed by CuSO4, which suggested that the inactivating factor(s) bound to the copper site of the enzyme. On a more rapid time scale than inactivation, papaya latex extract acted also as a weak noncompetitive PPO inhibitor.

Depletion of divalent cations within the secretory pathway inhibits the terminal glycosylation of complex carbohydrates of thyroglobulin

Newly synthesized thyroglobulin transiting the secretory pathway is posttranslationally modified by addition of oligosaccharides to asparagine N-linked residues. The effect of divalent cation depletion on oligosaccharide processing of Tg was studied in FRTL-5 cells. Treatment with an ionophore, A23187, or thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum ATPases delayed Tg secretion. These effects were accompanied by a normal distribution of the marker of the endoplasmic reticulum protein disulfide isomerase. Analysis of the thyroglobulin oligosaccharides by Bio-gel P4 chromatography showed that in the presence of A23187 and thapsigargin the addition of peripheral sialic acid and possibly galactose is inhibited. These findings were strengthened by experiments of exoglycosidase digestion and SDS-PAGE analysis of the resulting products. These results reveal a cellular mechanism of production of thyroglobulin with incompletely processed complex chains, i.e., the ligand of the recently described GlcNAc and asialoglycoprotein receptors of the thyroid. Since A23187 and thapsigargin inhibit biosynthetically the addition of peripheral sugars on N-linked oligosaccharides chains, the thyroglobulin molecules secreted in the presence of A23187 and thapsigargin should greatly facilitate studies on the function of the GlcNAc and asialoglycoprotein receptors of the thyroid.

Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods

Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolia lectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.

The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein.

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis (syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CR1SP-90) from a cloned Giardia isolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is posttranslationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by beta-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of[3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.

Purification of nonantibiotic insulinase inhibitors from bacitracin.

Bacitracin is commonly used in metabolic studies as an insulinase inhibitor. The many isoforms of the commercial preparation were fractionated by charge and size in order to find the most active rat-muscle insulinase inhibitors. CM-Sepharose chromatography revealed that most of the inhibitory activity was contained in a fraction (CM-Inh) that amounted to less than 5% of the mixture. The CM-Inh fraction could be further separated by size on Bio-Gel P4 columns. Six subgroups, each with characteristic specific activity, were isolated. The most potent inhibitor fractions have no antibiotic activity and have molecular weights about twice that of bacitracin A. The peaks isolated by means of Bio-Gel P4 chromatography can be further fractionated by reversed phase HPLC on a C8 column, and by electrophoresis on nonreducing acrylamide gels.

Complexity of ethanolamine phosphate addition in the biosynthesis of glycosylphosphatidylinositol anchors in mammalian cells.

Biosynthetic intermediates for the mammalian glycosylphosphatidylinositol (GPI) anchor have been described. The earliest GPI anchor precursor is N-acetylglucosaminylphosphatidylinositol, which is deacetylated to give glucosaminylphosphatidylinositol. This is followed by fatty acylation of the inositol ring, sequential addition of mannose residues donated by dolichyl mannosyl phosphate, and finally addition of ethanolamine phosphate. Here, we show that the final steps of GPI anchor biosynthesis are more complex than we have previously reported. Six distinct GPI anchor precursors were found to contain at least 1 ethanolamine phosphate residue. The headgroups of these glycolipids were purified and analyzed by a combination of Bio-Gel P4 chromatography and high resolution thin-layer chromatography. The sizes of neutral glycans were determined following HF dephosphorylation. The position of the ethanolamine phosphate residue was inferred from results of alpha-mannosidase treatment. Finally, the number of negative charges on the headgroups were determined by Mono Q chromatography. Our results show that the addition of ethanolamine phosphate is controlled by at least two different genes. Thus, the class F mutant, though unable to add ethanolamine phosphate to the third mannose residue, does incorporate ethanolamine phosphate into the first and second mannose residues. Only the wild type cells are capable of incorporating ethanolamine phosphate into the third mannose residue. Furthermore, the GPI core contains up to 3 ethanolamine phosphate residues. These results should facilitate the elucidation of the biochemical defects in paroxysmal nocturnal hemoglobinuria.

Characterization of O-linked oligosaccharide biosynthesis in cultured cells using paranitrophenyl α-D-GaINAc as an acceptor

Aryl-N-acetyl-alpha-galactosaminides (aryl-GalNAc) are acceptor substrates for UDP-Gal:alpha-GalNAc beta 1-3 galactosyltransferase and, in vivo, aryl-GalNAc have been shown to inhibit O-linked oligosaccharide biosynthesis (Kuan et al., J. Biol. Chem. 264, 19271, 1989). Since aryl-GalNAc, appears to enter viable cells and serve as an acceptor for O-glycosylation enzymes, the recovery and characterization of the aryl-oligosaccharides from cell culture medium may reflect cellular pattems of O-glycosylation. To pursue this possibility, the following paranitrophenyl-linked oligosaccharide standards were enzymatically synthesized and characterized by 1H-NMR: Gal beta 1-3(GlcNAc beta 1-6)Gal-NAc alpha-pNp; Gal beta 1-3(Gal beta 1-4GlcNAc beta 1-6)GalNAc alpha-pNp; SA alpha 2-3Gal beta 1-3(SA alpha 2-3Gal beta 1-4GlcNAc,beta 1-6)GalNAc alpha-pNp; SA alpha 2-3Gal beta 1-3GalNAc alpha-pNp. As a model system, MDAY-D2 lymphoid tumour cells were cultured for various periods in medium containing 2 mM GalNAc alpha-pNp. The secreted aryl-oligosaccharides were separated by Biogel P2 chromatography and DEAE HPLC, followed by further fractionation of the disialyl oligosaccharides on an Ultrahydrogel HPLC column. Absorbance of the paranitrophenyl aryl constituent at 303 nm allowed detection at the 10 pmol level and provided a relatively specific means of following the oligosaccharides. MDAY-D2 cells produced disialylated aryl-oligosaccharides at a rate of 20 pmol/h/10(6) cells with a half-time of transit to the cell surface of 13.6 min, a rate consistent with their movement from the Golgi to the cell surface by bulk flow.(ABSTRACT TRUNCATED AT 250 WORDS)

A Major Gibberellic Acid-Induced Barley Aleurone Cysteine Proteinase Which Digests Hordein

We previously described the purification and characterization of a 37,000 M(r) cysteine proteinase, designated EP-A, from gibberellic acid (GA(3))-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M(r) of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M(r) as determined by gradient SDS-PAGE.

The glycoinositolphospholipid profiles of two Leishmania major strains that differ in lipophosphoglycan expression

The glycolipid profiles of two Leishmania major strains which differ in their expression of the major glycoconjugate, lipophosphoglycan (LPG), have been compared. All the glycolipids in these strains belong to a class of glycoinositolphospholipids (GIPLs) which can be metabolically labelled with [3H]inositol and are sensitive to phosphatidylinositol-specific phospholipase C. The major glycolipids in the LPG-producing L. major strain V121 are tetraglycosyl phosphatidylinositol (GIPL-1), pentaglycosyl phosphatidylinositol (GIPL-2), hexaglycosyl phosphatidylinositol (GIPL-3) and lyso-GIPL-3. These were identified by their sensitivity to lipases, and by BioGel P4 chromatography of the glycan fragments released after nitrous acid deamination. Similar glycolipids are also present in the LPG-deficient L. major strain LRC-L119. However, this strain also produces several highly polar GIPLs (GIPL-4, 5 and 6) which are absent from V121 and which may represent truncated forms of LPG. Evidence is presented showing that the LPG and GIPLs from both strains contain galactofuranose, based on identification of labelled arabinose after mild periodate oxidation and reduction with NaB [3H]4. Furthermore, analysis of the deaminated glycan moieties after mild acid hydrolysis suggests that the GIPLs and the glycolipid anchor of LPG contain a common glycan core, which includes the galactofuranose. Finally, radiolabelling of intact cells indicates that there is restricted expression of some of the GIPLs in the plasma membrane and that the GIPL-2 is the predominant cell surface glycolipid. These data are consistent with some, but not all, of the GIPLs in V121 having a major role as precursors to the glycolipid core of LPG.

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins).

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.

Perfusion of uremic blood ultrafiltrate on uncoated charcoal.

A granular uncoated charcoal removes from uremic blood ultrafiltrates many chemical species that are not removed by dialysis. Charcoal treatment dramatically improves the general condition of the patients and normalizes their blood pressure. To obtain a rapid depuration, the initial treatment should be intensive (at least 16 daily treatments) and the effects prolonged over time by once-a-week charcoal treatment between two standard hemodialyses. Biogel P2 chromatography documents well all the events of a depurative treatment that cannot be monitored by hematochemical analyses.

A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase

A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [ 35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [ 35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [ 35S]cysteine, cysteine-protein adducts and [ 35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [ 35S]-tRNA was found to be the major reaction product, although some [ 35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.