Sequential Methylation Research Articles

Synthesis of methyl 3- [formula omitted]-[3- [formula omitted]-(2,3,4-tri- [formula omitted]-methyl-α-L-rhamnopyranosyl)-α-L-rhamnopyranosyl]-α-L-rhamnopyranoside : The outer trisaccharide unit of a unique [formula omitted][formula omitted] glycopeptidolipid

The combination of sugars present in Mycobacterium xenopi glycopeptido-lipid (GPL X-1) has been characterised as O -(2,3,4-tri- O -methyl-α- L-rhamnopyranosyl)-(1-3)- O -(α- L-rhamnopyranosyl)-(1-3) - O -(α- L-rhamnopyranosyl)-(1-3)-6-deoxy- L-glucose. Two synthetic routes (A and B) towards the outer trisaccharide segment (12) have been designed. Route A involved the condensation of the aglycone (7) with 2,3,4-tri- O -acetyl-α- L -rhamnopyranosyl bromide (13) to afford 8 which was sequentially deacetylated, methylated, deallylated and reacetylated to afford 12. Condensation of 12 and 16 in the presence of borontrifluoride etherate was unsuccessful. In route B, the disaccharide 21 was synthesised by the coupling reaction between 16 and 18 in the presence of borontrifluoride-etherate followed by deallylation. Alternatively, the disaccharide triacetate (22) was first prepared and routine modifications on it led to the formation of 21. Final condensation of 21 with the trichloroacetimidate (29) followed by sequential deacetylation, methylation and debenzylation afforded the trisaccharide (2).

Subunit-specific sulphation of oligosaccharides relating to charge-heterogeneity in porcine lutrophin isoforms.

Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of alpha and beta subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaB3H4, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO4-4GalNAc beta 1-4Glc-NAc and GalNAc beta 1-4GlcNAc or GlcNAc in S-1, SO4-4GalNAc beta 1-4GlcNAc and Sia alpha 2-6Gal beta 1-4GlcNAc in S-N, and (SO4-4GalNAc beta 1-4GlcNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and GlcNAc is N-acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the alpha and beta subunits were sulphated differently. S-1 predominated in the alpha subunit, while S-1 and S-2 were major components in the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylcholine biosynthesis in celery cell suspension cultures with altered sterol compositions

Cell suspension cultures of celery were treated with the plant growth regulator, paclobutrazol. Lipid analysis revealed that use of this xenobiotic had little effect on the quantity or acyl quality of the major phospholipid classes or on the actual amounts of free sterol present in the cell. It did however, cause dramatic changes in the free sterol profile exhibited by treated cultures. In this respect, an increase in 14α‐methylsterols was observed.The synthesis of phosphatidylcholine in celery cell suspension cultures with altered free sterol compositions was studied using two radiolabelled biosynthetic precursors, [3H‐methyl]choline and [3H‐methyl]methionine. The studies showed that the rate of phosphatidylcholine biosynthesis via the CDP‐base pathway proceeded at a slower rate in paclobutrazol treated cultures. Accumulation of label phosphocholine was observed arising from reduced CTP:cholinephosphatecytidylyltransferase activity. In contrast, phosphatidylcholine biosynthesis via the sequential methylation of ethanolamine derivatives appeared to be enhanced in cells that had an unusually high 14α‐methylsterol content. From these investigations it may be postulated that the biosynthesis of phosphatidylcholine in Apium graveolens suspension cultures may be regulated by membrane sterol composition.

Effect of Monomethylethanolamine, Dimethylethanolamine, Gangliosides, Isoproterenol, and 2‐Hydroxyethylhydrazine on the Conversion of Ethanolamine to Methylated Products by Cultured Chick Brain Neurons

The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10(-8) M and 10(-5) M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10(-6) M and 10(-5) M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 x 10(-5) M propranolol.

Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells.

Cultured adrenal chromaffin cells, representing a virtually homogeneous population of neuronal elements, have been utilized to examine the final enzymes in the formation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), namely, choline phosphotransferase, ethanolaminephosphotransferase, and the N-methyltransferases in the sequential methylation of PE to PC. Each enzyme has been characterized extensively in terms of substrate requirements, pH optima, detergent and cation effects, and response to inhibitors revealing properties very similar to those in other neural preparations. The respective activities are stable for up to two weeks of adrenal chromaffin cell culture suggesting that this system is a suitable model for examining the relative roles and the regulation of each pathway in PC formation.

A disialoganglioside of the globo-series from chicken skeletal muscle

We have isolated a disialoganglioside of the globo-series from chicken pectoral muscle. The compound was obtained by extraction followed by ion-exchange and silicic acid column chromatography and judged to be pure by thin-layer chromatography in three solvent systems. The structure of the ganglioside was determined by carbohydrate and ceramide composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-MHz 1H-NMR spectroscopy to be: (formula; see text) Analysis of the ceramide moiety indicated d18:1 sphingosine as the long-chain base, and C16:0, C18:0, C18:1, and C20:0 as the prevalent fatty acids. This glycolipid is only the second ganglioside of the globo-series, and the first disialo member of the series, found in chicken muscle.

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin from a patient with extragonadal germ cell tumour

Human chorionic gonadotropin (hCG) purified from the urine of a male patient with extragonadal germ cell tumour contained four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively released from the polypeptide moiety by hydrazinolysis and recovered as oligosaccharides after N-acetylation. The oligosaccharide mixture was separated into a neutral (N) and three acidic (A1, A2 and A3) fractions by anion-exchange column chromatography. By sequential exoglycosidase digestion, methylation analysis and lectin column chromatography, the structures of these oligosaccharides were found to be the same as those of female gestational choriocarcinoma hCGs. Both contain eight kinds of sugar chains: triantennary, abnormal and normal biantennary, and monoantennary complex-type sugar chains with or without a fucosylated core portion.

Elevation of the activities of glycosyl transferases involved in polylactosaminoglycan biosynthesis in autoimmune MRL lpr/ lpr mouse T cells

Lymph node (LN) T cells from autoimmune MRL/MpJ- lpr/ lpr (lpr) mice and control MRL/MpJ- + / + ( + / + ) mice were compared as to glycosyl transferase activities involved in the biosynthesis of polylactosaminoglycans. The N-acetylglucosaminyl transferase (GlcNAc transferase) activity responsible for the extension of polylactosaminoglycans was assayed. The reaction products with this GlcNAc transferase were characterized by sequential glycosidase treatment and methylation analysis, and the enzyme was found to be classifiable as an UDP-GlcNAc: N-acetyllactosamine β1–3 GlcNAc transferase (polylactosamine extension enzyme). The activity of this GlcNAc transferase in T cells from enlarged LN of Ipr mice was 3–6 times higher than that in T cells from + / + mice. On the other hand, activated T cells from + / + mice only showed about a 2-fold increase in the activity of the transferase, compared with that in resting T cells. B cells from + / + mice also showed a significantly higher activity of the transferase than + / + T cells, the enzyme activity being comparable to or slightly lower than that in lpr T cells. Furthermore, when the reaction mixture contained both UDP-GlcNAc and UDP-Gal as donors, extension of the Gal-GlcNAc residue was observed. These results indicated the biosynthetic basis for the abundance of polylactosaminoglycans in lpr T cells and normal B cells. We also found that lpr T cells exhibited significant UDP-GlcNAc:asialo-bovine submaxillary mucin GlcNAc transferase activity. Only weak activity of this enzyme was detected in + / + resting and activated T cells, and B cells. This enzyme activity suggested the potential for polylactosaminoglycan formation on the mucin-type sugar chains on the surface of lpr T cells.

Chromium Mediated Organic Synthesis: The Introduction of 1,1-Dimethylallyl and Linalyl Groups on to Arenes

The synthesis of 1,1-dimethylallyl-substituted benzene-and anisoletricarbonylchromium(0) complexes was achieved in a two- or three-step sequence by the copper catalysed coupling of the lithiated arenetricarbonylchromium(0) complexes with allyl bromide followed by a double benzylic alkylation with potassium tert-butoxide/methyl iodide. For the linalyl substituted complexes the alkylations were sequential homoprenylation and methylation. Alternatively, palladium catalysed cross coupling with 2-bromo-2-butene followed by γ-deprotonation of the 1-aryl-1-propene complexes provided a more direct approach to the anionic intermediates involved.

Total synthesis of (+)-albicanol and (+)-albicanyl acetate via a highly diastereoselective intramolecular [3 + 2] cycloaddition

The enantioselective total syntheses of the drimane-type sesquiterpenes albicanol (1) and albicanyl acetate(2) starting from (+)-Wieland–Miescher ketone (8) have been accomplished. The key step in the synthetic strategy involves a highly diastereoselective intramolecular [3 + 2] dipolar cycloaddition reaction of the nitrile oxide (6), which affords the isoxazoline (5) as the sole product in high yield. Reductive hydrolysis of compound (5) followed by methylenation of the resulting β-hydroxy ketone (4) by application of the Nozaki–Lombardo procedure provides (+)-albicanol (1), which is then converted into (+)-albicanyl acetate (2) by acetylation. The olefinic acetal (15), an intermediate of the present total synthesis, has been transformed by sequential Jones oxidation and methylation into a secosesquiterpene (17), one of the components of sun-cured Greek tobacco.

Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.

Purification and Characterization of the Epitectin from Human Laryngeal Carcinoma Cells

The purification and partial characterization of epitectin (previously called Ca antigen) from a human cancer cell line is described. This glycoprotein, which is expressed on a wide range of human tumors and certain specialized normal epithelia, can be detected using monoclonal antibodies, Ca1, Ca2, and Ca3. The purified glycoprotein had a high density (1.40 g/ml) on isopycnic centrifugation indicating a high carbohydrate content. The molecular mass of epitectin as determined by size-exclusion chromatography ranged from 1.0 to 1.5 x 10(6) daltons. However, the purified epitectin gave two bands of apparent molecular weight 390,000 and 350,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of epitectin and asialoepitectin were found to be 5.3-5.4 and 6.8, respectively. The oligosaccharides were isolated from metabolically labeled epitectin by alkaline borohydride treatment and their structures established based on high performance liquid chromatography and paper electrophoretic migration, sugar composition, the results of sequential exoglycosidase treatment, periodate oxidation, and methylation analysis. The structures of the three major fractions, which together account for about 80% of the radioactivity, were assigned as NeuNAc alpha 2----3Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----3Gal beta 1----3GalNAc(OH), and Gal beta 1----3 GalNAc(OH). The structures of the minor fractions were tentatively assigned as NeuNAc----Gal(NeuNAc----Gal----GlcNAc)----GalNAc(OH), Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----6GalNAc(OH), and GalNAc(OH). It is proposed that the protein sequence and/or the distribution of the saccharides on the protein core are the determinants on epitectin that are recognized by the Ca antibodies.

Structural study of the carbohydrate moieties of two human immunoglobulin subclasses (IgG2 and IgG4)

Asparagine-linked sugar chains were quantitatively released as oligosaccharides from human IgG2 and IgG4 myeloma proteins by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Each oligosaccharide was isolated by serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion and methylation analysis, revealed that all of them were of the bi-antennary complex-type containing Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc as core structures, and GlcNAc beta 1-, Gal beta 1-4GlcNAc beta 1- and Sia alpha 2-6Gal beta 1- in their outer chain moieties. However, the molar ratio of each oligosaccharide was different in each IgG sample, indicating that clonal variation is included in the sugar chain moieties of IgG molecules. One of the IgG2 contained four asparagine-linked sugar chains in one molecule, two on the Fc fragment and the remainder on the Fab fragment. The sugar chains in the Fc fragment contained much less galactose as compared with the Fab fragment.

Rat and human mammary tissue can synthesize choline moiety via the methylation of phosphatidylethanolamine.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.

The 5′-GGATCC-3′ cleavage specificity of BamHl is increased to 5′-CCGGATCCGG-3′ by sequential double methylation with M · HpaII and M · BamHI

Site-specific DNA methylation is known to block cleavage by a number of restriction endonucleases. We show that methylation at ‘non-canonical’ DNA modification sites can also block methylation by five of 13 DNA methyltransferases (MTases) tested. Furthermore, MTases and endonucleases that recognize the same nucleotide sequence can differ in their sensitivity to non-canonical methylation. In particular, BamHI endonuclease can cut 5′-GGATCm 5C efficiently, whereas M· BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites. For example, we show that M· HpaII, M· BamHI, and BamHI can be used sequentially in a three-step procedure to specifically cleave DNA at the 10-bp sequence 5′-CCGGATCCGG. Several highly selective DNA cutting strategies are made possible by these sequential double methylation-blocking reactions.

Comparative studies of the sugar chains of aminopeptidase N and dipeptidylpeptidase IV purified from rat kidney brush-border membrane

Asparagine-linked sugar chains of rat kidney aminopeptidase N and dipeptidylpeptidase IV were investigated comparatively. Oligosaccharides released from the two enzymes by hydrazinolysis were fractionated by paper electrophoresis, serial chromatographies on columns of immobilized Aleuria aurantia lectin, concanavalin A, phytohemagglutinin E4, and Datura stramonium agglutinin, and Bio-Gel P-4 (less than 400 mesh) column chromatography. Structures of oligosaccharides in each fraction were assumed by their effective molecular sizes and behaviors on the four lectin columns and then confirmed by sequential exoglycosidase digestion and methylation analysis. The sugar chains of aminopeptidase N and dipeptidylpeptidase IV are almost the same as those of rat kidney gamma-glutamyltranspeptidase reported previously [Yamashita, K., Hitoi, A., Matsuda, Y., Tsuji, A., Katunuma, N., & Kobata, A. (1983) J. Biol. Chem. 258, 1098-1107]. They are a mixture of 5 high mannose type sugar chains and 32 (for aminopeptidase N) or 26 (for dipeptidylpeptidase IV) mono-, bi-, tri-, and tetraantennary complex type sugar chains. The unique feature of the complex-type sugar chains of both enzymes is that they all contain the bisecting N-acetylglucosamine residue and are incompletely galactosylated in their outer-chain moieties.

Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked d-glucopyranosyluronic residues

The fate of 4-linked d-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra- O-methyl- d-glucitol, 3,4-di- O-acetyl-1,5-anhydro-2,6-di- O-methyl- d-glucitol, and 3- O-acetyl-1,5- anhydro-2,4-di- O-methyl- l-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked d-glucopyranosyluronic residue, namely, methyl 3- O- acetyl-2,6-anhydro-4,5-di- O-methyl- l-gulonate, was not observed. Instead, methyl 2- O-acetyl-3,6-anhydro-4,5-di- O-methyl- l-gulonate ( 6) was identified as the sole product of reductive cleavage of the 4-linked d-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked d-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di- O-acetyl-1,5- anhydro-2,3-di- O-methyl- d-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked- d-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked d-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

Structural study on the carbohydrate moiety of human placental alkaline phosphatase.

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.

Arsenate metabolism in aquatic plants

AbstractOf the several types of arsenic metabolic pathways in algae and aquatic higher plants, production of arsenoribosides is predominant and most interesting. Insertion of the ribosyl or adenosyl moiety by transfer from S‐adenosylmethioninine or possibly from adenosylcobalamin is the critical biochemical step which, as yet, has not been experimentally demonstrated. Oceanic or other environmental arsenate (AsO43−), absorbed in the plant's quest for phosphate, is fixed by reaction with ATP to yield the phosphoric arsenic anhydride APAs, a short‐lived but reducible intermediate which is converted to arsenic(III). The Hill Reaction, or thioredoxin reductase, reduces it to an arsine oxide (HAsO) or arsonous acid [HAs(OH)2] depending upon the water content of its environment. This readily diffusible reagent avidly attacks sulfhydryl groups of proteins to produce arsonous thioesters. The Sargassum group of algae appears to process arsenate no further than this. The reduced arsenic may be freed from its sulfur bondage by reaction with 2,3‐dimercaptopropan‐1‐ol (BAL) or dithiothreitol. In experiments with Sargassum fluitans and Sargassum natans no arsenoribosides were observed. Only protein‐bound arsenic was observed. It could be liberated by trituration with dithiothreitol to produce the cyclic arsonous dithioester.Most diatoms, dinoflagellates and macroalgae as well as freshwater higher plants release such protein‐bound arsenic as a result of sequential methylation and adenosylation. Ultimately the products are trialkylarsine oxides, innocuous substances which are slowly or not‐at‐all metabolized by herbivorous animals or bacteria. Fortunately mammals and most animals also excrete the arsenoribosides readily, unchanged. Arsenic metabolism by a cyanobacterium, Phormidium sp., was described by Matsuto et al. (Comp. Biochem. Physiol., 1984, 78c:377) as involving two modes of arsenate fixation, reduction, and excretion. We have extended those experiments with Phormidium persicinum. We have analyzed algae from 2‐ and 7‐day culture in radioarsenate media. The arsenic products included 80% of arsenolipid, similar if not identical to that formed in the brown algae. The water‐soluble products were in low concentration. Insoluble, protein‐bound arsonous thioesters accounted for 8% of the fixed arsenic. The mechanism of arsenic depuration in Phormidium appears to be primarily lipid‐mediated.

Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line.

The carbohydrate structure of the major oligosaccharide of human interferon-beta (IFN-beta) synthesized by a genetically engineered Chinese hamster ovary cell line has been determined. Analysis of the glycopeptidase F-released carbohydrates by sequential exoglycosidase treatment, methylation analysis, and fast atom bombardment-mass spectrometry revealed that 95% of the IFN-beta oligosaccharides had the following structure: (Formula: see text). The remaining 5% of the carbohydrates are probably tri- or higher antennary oligosaccharide chains. The major oligosaccharide of the recombinant IFN-beta is remarkably homogeneous with respect to terminal galactose sialylation. NeuAc, which is alpha 2-3-linked to galactose in the human IFN-beta secreted by Chinese hamster ovary cells, can be re-incorporated with an alpha 2-6 linkage in vitro, into enzymatically desialylated IFN-beta using rat liver Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. The sugar chain is important for maintaining protein solubility as shown by the fact that IFN-beta protein precipitates after deglycosylation with glycopeptidase F.