Similar Carbohydrate Compositions Research Articles

Sustainable Exopolysaccharide Production by Rhizobium viscosum CECT908 Using Corn Steep Liquor and Sugarcane Molasses as Sole Substrates.

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by Rhizobium viscosum CECT908, containing sugarcane molasses (60 g/L) and corn steep liquor (10 mL/L) as sole ingredients. This medium allowed the production of 6.1 ± 0.2 g EPS/L, twice the amount produced in the standard medium (Syn), whose main ingredients were glucose and yeast extract. This is the first report of EPS production by R. viscosum using agro-industrial residues as sole substrates. EPSCSLM and EPSSyn exhibited a similar carbohydrate composition, mainly 4-linked galactose, glucose and mannuronic acid. Although both EPS showed a good fit to the Herschel-Bulkley model, EPSCSLM displayed a higher yield stress and flow consistency index when compared with EPSSyn, due to its higher apparent viscosity. EPSCSLM demonstrated its potential use in Microbial Enhanced Oil Recovery by enabling the recovery of nearly 50% of the trapped oil in sand-pack column experiments using a heavy crude oil.

INFLUENCE OF HYDROLYTIC TREATMENT ON THE CONTENT OF REDUCING SUBSTANCES IN NEUTRAL-SULPHITE ALKALI

The aim of this work is to develop a technology for the preparation of neutral-sulfite liquors formed during the production of fibrous semi-finished products - cellulose from birch wood - for subsequent use as a nutrient medium for the cultivation of microorganisms. Acid hydrolysis was carried out at a temperature of 100 °С at a ratio of a 10% sulfuric acid solution to a liquor sample of 1 : 1. Enzymatic hydrolysis of neutral sulfite liquors was carried out with the enzyme preparations Accellerase XY and Accellerase XC at 50±2 °C and 60±2 °C. The end of hydrolysis was determined by the cessation of the increase in the content of reducing substances (RS) in the hydrolyzate. The original neutral sulphite lye contained 9.4% dry matter, 21.7 g/l of reducing substances, pH 5.3±0.2. It has been shown that as a result of enzymatic hydrolysis, the content of insoluble dry residue in the hydrolyzate decreases to 8.32% and 8.41%, respectively, and during acid hydrolysis – to 7.8%. The content of RS in neutral sulfite lye after acid hydrolysis increases by an average of 3 times, while after enzymatic hydrolysis - a maximum of 2 times. It was found by gas-liquid chromatography that pentoses predominate in the obtained hydrolysates. Microbiological processing of media with a similar carbohydrate composition is possible by a number of strains of microorganisms capable of assimilating pentoses, for example, yeast-like fungi of the Saccharomycetaceae family and bacteria of the Enterobacteriaceae family.

Comparison of saccharification and fermentation of steam exploded rice straw and rice husk.

BackgroundRice cultivation produces two waste streams, straw and husk, which could be exploited more effectively. Chemical pretreatment studies using rice residues have largely focussed on straw exploitation alone, and often at low substrate concentrations. Moreover, it is currently not known how rice husk, the more recalcitrant residue, responds to steam explosion without the addition of chemicals.ResultsThe aim of this study has been to systematically compare the effects of steam explosion severity on the enzymatic saccharification and simultaneous saccharification and fermentation of rice straw and husk produced from a variety widely grown in Vietnam (Oryza sativa, cv. KhangDan18). Rice straw and husk were steam exploded (180–230 °C for 10 min) into hot water and washed to remove fermentation inhibitors. In both cases, pretreatment at 210 °C and above removed most of the noncellulosic sugars. Prolonged saccharification at high cellulase doses showed that rice straw could be saccharified most effectively after steam explosion at 210 °C for 10 min. In contrast, rice husk required more severe pretreatment conditions (220 °C for 10 min), and achieved a much lower yield (75 %), even at optimal conditions. Rice husk also required a higher cellulase dose for optimal saccharification (10 instead of 6 FPU/g DM). Hemicellulase addition failed to improve saccharification. Small pilot scale saccharification at 20 % (w/v) substrate loading in a 10 L high torque bioreactor resulted in similarly high glucose yields for straw (reaching 9 % w/v), but much less for husk. Simultaneous saccharification and fermentation under optimal pretreatment and saccharification conditions showed similar trends, but the ethanol yield from the rice husk was less than 40 % of the theoretical yield.ConclusionsDespite having similar carbohydrate compositions, pretreated rice husk is much less amenable to saccharification than pretreated rice straw. This is likely to attenuate its use as a biorefinery feedstock unless improvements can be made either in the feedstock through breeding and/or modern biotechnology, or in the pretreatment through the employment of improved or alternative technologies. Physiological differences in the overall chemistry or structure may provide clues to the nature of lignocellulosic recalcitrance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0599-6) contains supplementary material, which is available to authorized users.

Influence of Biomass Chemical Properties on Torrefaction Characteristics

Different biomass types may differ with respect to torrefaction characteristics, and an improved understanding and ability to predict the torrefaction performance is, therefore, desired. In this study, the influence of the chemical properties (lignocellulose composition and alkali content) on the torrefaction behavior with respect to mass loss and grindability is investigated by simultaneous thermal analysis (STA) and by using a combined torrefaction and grinding reactor. The torrefaction behavior of six raw biomass samples and selected pretreated samples (washed and impregnated with KCl and K2CO3) has been studied. The investigated biomasses have reasonably similar carbohydrate compositions (hemicelluloses 18–25 wt % db; cellulose 38–48 wt % db; lignin 17–29 wt % db) with the exception of spruce bark, which is lower in hemicellulose content (12.9 wt % db) and cellulose content (24 wt % db), and higher in lignin content (36.8 wt % db). An increasing biomass potassium (K) content decreases the temperature of maximal conversion for both raw and alkali-impregnated biomass samples, thus decreasing the solid product yield at 270 and 300 °C. This was especially pronounced when the biomass potassium content increased from 0 to 0.2 wt %. However, the higher lignin content in bark causes a higher solid yield than what would be expected from the alkali content, illustrating that both potassium content and lignocellulose composition affect the solid yield obtained by torrefaction. The grindability of the torrefied products was evaluated by determining the d50 value of the particle size distribution of the biomass before and after torrefaction in the combined torrefaction and mill reactor. A significant decrease in d50 value was observed when the alkali content increased from 0 to 0.2 wt % db, whereas no additional effect is seen for higher potassium contents.

Effects of high-fat and low-fat diets rich in monounsaturated fatty acids on serum lipids, LDL size and indices of lipid peroxidation in healthy non-obese men and women when consumed under controlled conditions

To study the effects of the dietary fat content on cardiovascular disease risk factors in humans when the fatty acid composition and types of carbohydrates are kept constant. A controlled dietary study in healthy volunteers with 2 dietary groups and a parallel design consisting of 2 dietary periods was conducted. First, participants received a 2-week wash-in diet rich in saturated fatty acids (SFA; 47% of total fatty acids) and were then randomly assigned to either a high-fat (40% of energy) or a low-fat diet (29% of energy) for 4 weeks. Both diets were isocaloric, rich in monounsaturated fatty acids (MUFA; 51% of total fatty acids) and had similar fatty acid and carbohydrate compositions. Compared to the wash-in diet, the high-fat and low-fat diets significantly lowered LDL-cholesterol (-0.34 and -0.41 mmol/l, respectively; P < 0.001 for time effect in RM-ANOVA), and HDL-cholesterol (-0.13 and -0.18 mmol/l, respectively; P < 0.001 for time), without any differences between the high-fat and low-fat diets (P = 0.112 and P = 0.085 for time × group interaction in RM-ANOVA, respectively). The size of the major LDL fraction, the LDL susceptibility to oxidation and the plasma concentrations of oxidized LDL (ox-LDL) were significantly reduced by both the high-fat and low-fat diet, again without significant differences between the diets. The ratio of ox-LDL/LDL-cholesterol, serum triacylglycerols and urinary F2-isoprostanes were not significantly affected by the diets. A high-fat and a low-fat diet, both rich in MUFA, had similar effects on lipid-related cardiovascular disease risk factors in metabolically healthy men and women.

Seminal fluid from sea urchin (Lytechinus variegatus) contains complex sulfated polysaccharides linked to protein

The eggs of sea urchins are covered by a jelly coat, which contains high concentrations of sulfated polysaccharides. These carbohydrates show species-specificity in inducing the sperm acrosome reaction. Several studies about the egg jelly of sea urchins have been published, but there is no information about the composition of the seminal fluid of these echinoderms. Here we report for the first time the occurrence of complex sulfated polysaccharides in the seminal fluid of the sea urchin Lytechinus variegatus. These polysaccharides occur as three fractions that differ mostly in their carbohydrate/protein ratios. The native molecular masses of the polymers are very high (> or = 200 kDa) but, after digestion with papain the size decreases to approximately 8 kDa. All fractions have a similar carbohydrate composition, containing mostly galactose, glucosamine and mannose. The heterogeneous sulfated polysaccharides differ from vertebrate glycosaminoglycans and also from all previously described polysaccharides from invertebrates. The physiological role of the sulfated carbohydrates from seminal fluid is not yet determined. However, by analogy with the effects proposed for some glycoproteins found in vertebrate seminal fluid, it may be possible that the sulfated polysaccharides from invertebrate are also involved in fertilization process.

Cross polarisation/magic angle spinning 13C-NMR spectroscopic studies of cellulose structural changes in hardwood dissolving pulp process

Abstract Cross polarisation/magic angle spinning 13C NMR spectroscopy has been used to study structural changes in cellulose induced by the dissolving pulp process. The cellulose structure in several dissolving pulps was investigated for commercial and laboratory cooked Eucalyptus 92α and 96α. The average lateral dimension, or average thickness, of the cellulose fibril aggregates is related to the amount of surface area exposed and could be one controlling factor for the chemical reactivity of commercial dissolving pulps during modification reactions. The thickness of the cellulose fibril aggregates governs the amount of surface area present in the fibre wall, and cellulose surface material constitutes the part of the cellulose that is directly accessible to reagents. In all sample series investigated, the raw pulp was found to be less aggregated than the corresponding bleached final pulp. Furthermore, an irreversible increase in fibril aggregate width was observed on free drying for both laboratory cooked and commercial pulps. Upon rewetting with water, the freely dried 96α pulp was found to be more aggregated than the freely dried 92α pulp, although sugar analysis showed very similar carbohydrate compositions. As indicated by the molecular mass distribution, the commercial 92α pulp contained larger amounts of degraded cellulose; this may be a plausible explanation for the different behaviour of the 92α and 96α pulps during free drying.

Novel elicitin‐like proteins isolated from the cell wall of the biocontrol agent Pythium oligandrum induce defence‐related genes in sugar beet

SUMMARY We previously reported that cell wall protein fractions (CWPs) of the biocontrol agent Pythium oligandrum have elicitor properties in sugar beet and wheat. Here we have examined the effect of treatment with the D-type of CWP, a fraction that contains two major forms (POD-1 and POD-2), on the induction of defence-related genes in sugar beet. Using PCR-based cDNA library subtraction, we identified five genes that were highly expressed in response to CWP treatment. The five genes are probably of oxalate oxidase-like germin (OxOLG), glutathione S-transferase (GST), 5-enol-pyruvylshikimate-phosphate synthase (EPSPS), phenylalanine ammonia-lyase (PAL) and aspartate aminotransferase (AAT). In addition, we purified and characterized POD-1 and POD-2 and found that POD-1 induced all five genes, whereas POD-2 induced three of the genes, but not OxOLG or GST. A sugar beet bioassay indicated that CWP, POD-1 and POD-2 are each sufficient to induce resistance to sugar beet seedling disease caused by Aphanomyces cochlioides. Although carbohydrate analyses indicated that POD proteins were glycoproteins with similar carbohydrate compositions, containing approximately 15.0% carbohydrate by weight, their peptide portions have elicitor activity. Furthermore, cDNAs of POD-1 and POD-2 proteins were cloned, and the deduced amino acid sequences were found to be 82.9% identical. Characterization of their molecular structures indicated that they have an elicitin domain followed by a C-terminal domain with a high frequency of Ser, Thr, Ala and Pro, which is structurally similar to class III elicitins. However, phylogenetic analysis with 22 representative elicitin and elicitin-like proteins showed that POD-1 and POD-2 are distinct from previously defined elicitin and elicitin-like proteins. Therefore, POD-1 and POD-2 are novel oomycete cell wall elicitin-like glycoproteins.

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as α-2,6-linkage and no α-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.

Peripartal Metabolism and Production of Holstein Cows Fed Diets Supplemented with Fat During the Dry Period

Previous research from our laboratory demonstrated that cows fed supplemental fat throughout the dry period in an attempt to increase body condition score (BCS) had little hepatic lipid accumulation at d 1 postpartum compared with cows fed an isocaloric high-grain diet or a lower energy control diet. However, results were confounded by lower dry matter intake and loss of BCS by cows fed the fat-supplemented diet. Here, cows were fed a control diet (C) moderately high in nonfiber carbohydrates (NFC) or an isocaloric fat-supplemented, low NFC (F) diet to reassess the effects of supplemental fat throughout the dry period on peripartal lipid accumulation in liver. A more energy-dense, high-NFC diet supplemented with fat (CF) was also fed to test the efficacy of supplemental fat in a diet with similar carbohydrate composition but higher energy density. Intakes of dry matter and net energy for lactation were similar among treatments throughout the experiment, although diet×day interactions during the last 21 d before parturition indicated that cows fed CF decreased intakes more slowly. Cows gained similar amounts of BCS and body weight among diets prepartum, but cows fed C tended to lose more BCS and body weight around parturition. Milk production and milk components did not differ among treatments. Prepartum concentrations of glucose, insulin, total protein, nonesterified fatty acids, and μ-hydroxybutyrate in plasma were similar among treatments. Supplemental fat increased prepartum concentrations of urea and cholesterol in plasma. Postpartum concentrations of metabolites and insulin in plasma were similar among treatments. Concentrations of total lipid and triglyceride in liver increased at parturition, whereas hepatic glycogen concentration decreased, but concentrations were not different among treatments. Supplemental fat fed prepartum did not affect peripartal lipid accumulation in liver tissue and did not benefit postpartum milk production.

Isolation and Characterization of Glycophorin from Nucleated (Chicken) Erythrocytes

A sialoglycoprotein fraction was isolated from chicken erythrocytes by two methods based on the phenol extraction or chloroform/2-propanol extraction of differently prepared erythrocyte membranes. Both preparations gave in SDS–PAGE two major PAS-stained bands (GP2 and GP3), which migrated as 60- and 33-kDa species, respectively, compared to reference proteins, or as 44- and 23-kDa molecules, compared to human glycophorins. Some less abundant slower migrating PAS-stained components, antigenically related to GP2 and GP3, also were detected. No evidence for the presence of antigenically distinct glycoproteins of leukosialin type was obtained. Interconversion in SDS–PAGE, similar carbohydrate composition, and similar antigenic properties of GP2 and GP3 indicated that they are a dimer and monomer, respectively, of the same glycoprotein which shows properties that allow it to be classified as a glycophorin. Lectin binding studies and methylation analysis of β-elimination products of chicken glycophorin preparation showed the presence of O-glycans and N-glycans. The major O-glycans include sialylated Galβ1–3GalNAc units and more complex GlcNAc-containing chains. Among the N-glycans, there are complex-type biantennary structures with a bisecting GlcNAc residue, accompanied by chains with additional antennas linked to α-mannose residues. A characteristic feature of the chicken glycophorin is a relatively high proportion of N-glycans to O-glycans, compared to the glycophorin A from human erythrocytes.

Composition of cell walls isolated from cell types of grain sorghum stems

Cell types were isolated from sorghum stems at two stages of development, anthesis and grain maturity, to study cell wall characteristics. Cell walls were isolated from epidermis (EPID), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) and analysed for total carbohydrate, acid insoluble lignin, total uronosyls, neutral sugars and hydroxycinnamic acids. In addition, walls from SCL, VBZ, IVB and PITH were subjected to chemical fractionation to separate wall carbohydrate into polysaccharide groups. Although wall characteristics were similar at both plant maturities, there were differences in lignin concentration, hydroxycinnamic acids, and carbohydrate composition among the cell wall types. Lignin was lowest in the PITH walls (169 g kg−1) and highest in SCL and EPID (c 211 g kg−1). Cellulose was most abundant in VBZ and SCL walls with greater secondary wall formation. Pectic materials were most abundant in PITH walls. Xylans were similar among wall types except for EPID that contained higher amounts of xylose. Releasable hydroxycinnamates were not as consistent among the cell wall types. Total ferulates, including ester linked and releasable ether linked, tended to increase from PITH to SCL (8 to 15 g kg−1 CW) with an increase in the proportion etherified within the wall matrices (PITH 51%; SCL 66%). Total p-coumarates showed opposite trends with PITH walls having significantly more (35 g kg−1 CW) than VBZ or SCL (19 and 13 g kg−1 CW). EPID walls contained the least pCA (6.5 g kg−1 CW). Except for the hydroxycinnamates, compositional trends for the different wall types would reflect changes from primary walls to increased amounts of secondary wall. Neutral sugar analysis of indigestible residues indicated similar carbohydrate compositions among the cell wall types, with xylose being less degradable than all other wall sugars. © 1999 Society of Chemical Industry

Effects of synchronizing the rate of dietary energy and nitrogen release in diets with a similar carbohydrate composition on rumen fermentation and microbial protein synthesis in sheep

SUMMARYThe effects of synchronizing dietary energy and nitrogen supply in diets with a similar carbohydrate composition on microbial protein synthesis and rumen fermentation were examined in sheep. Two diets were formulated to be either synchronous (diet S) or asynchronous (diet A) for the hourly release of nitrogen (N) and energy to the rumen. Diet S contained (g/kg) 425 g wheat straw, 400 g winter barley, 150 grapeseed meal and 25 g minerals/vitamins and diet A contained 505 g wheat straw, 458.5 g winter barley, 11·5 g urea and 25 g minerals/vitamins. Both diets were fed at the rate of 1 kg/day in four equal portions, to four cannulated sheep, in two periods in a change-over design. Rumen ammonia concentrations followed the predicted hourly trend in N degradation with a peak 1 h after feeding of 10 mM for diet S and 16 mM for diet A before falling within 3 h of feeding to 4 ITIM in animals fed either diet. Rumen volatile fatty acid (VFA) concentrations followed the cyclical trend predicted by stoichiometric equations, whilst rumen VFA ratios were more stable than predicted in animals fed either diet. The observed content of rumen degradable protein and organic matter truly degraded in the rumen was similar for both diets. The increase in total CHO digested in the rumen observed with diet A (427 g/kg DM) compared with diet S (364 g/kg DM) can be attributed to the greater content of starch in the asynchronous diet, which had a high degradability. The efficiency of microbial protein synthesis (g N/kg OM truly degraded in the rumen) was 11–20% greater in animals fed the synchronous diet (S) than the asynchronous diet (A). It is concluded that microbial N production was more efficient when dietary energy and N supply were synchronized.

The molecular characterisation of the polysaccharide gum from Acacia senegal

Gum arabic ( Acacia senegal gum) was fractionated by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B and yielded four fractions of similar carbohydrate composition, whilst differing in protein content, amino acid composition, and molecular mass distribution. All four fractions interacted with an array of anti-arabinogalactan-protein (AGP) monoclonal antibodies via anti-carbohydrate epitopes and were precipitated by Yariv's reagent, suggesting that they were all AGPs. Further characterisation using SDS-PAGE and Western blotting showed that all fractions contained proteins of molecular mass 30 000–45 000 with at least one protein unique to each fraction. The fractions were subjected to enzymatic (with pronase) and alkaline hydrolysis, and the molecular mass distribution of the products was monitored by gel permeation chromatography. The fractions showed various degrees of resistance to hydrolysis, thus illustrating differences in their macromolecular structure.

Effects of synchronising dietary nitrogen and energy supply in diets with a similar carbohydrate composition on rumen fermentation and microbial protein synthesis in sheep

The recently introduced metabolisable protein system for ruminants (Webster 1992) relates microbial nitrogen production to daily supply of fermentable metabolisable energy and effective rumen degradable protein but does not consider the effect of the pattern of supply of nutrients to rumen microbes on their efficiency and growth. However, synchronising the hourly supply of nitrogen and energy yielding substrates to rumen micro-organisms has been shown to increase the efficiency of microbial protein synthesis (Sinclair et al. 1993). The objective of the current experiment was to examine the effects of synchronising the hourly supply of energy and nitrogen in diets with a similar carbohydrate composition but differing in the rate of protein degradation, on rumen fermentation and microbial protein synthesis in sheep.

Biochemical properties of tracheobronchial mucins from cystic fibrosis and non-cystic fibrosis individuals.

Tracheobronchial mucins from healthy individuals and from patients with bronchial asthma or cystic fibrosis (CF) were isolated from lung mucus, purified, and their chemical and physical properties compared. Normal and asthmatic mucins required both a dissociating and a reducing agent for solubilization and exhibited identical chromatographic behavior on Sepharose 4B, Sepharose 2B, and hydroxylapatite and similar amino acid and carbohydrate compositions. In contrast, 1) CF lung mucins were solubilized in the absence of dissociating and/or reducing agents and 2) the majority of the CF mucins analyzed was eluted in the included volume of Sepharose 4B with Kd values of 0.3 +/- 0.1 rather than in the void volume and thus appeared smaller than normal and asthmatic mucins. The lower molecular weight mucins in CF sputum apparently are produced by bacterial or inflammatory cell proteinases since radiolabelled asthmatic mucin was digested to smaller fragments when incubated with crude CF lung mucosal samples. Furthermore, mucins secreted by tracheal explants from CF and from non-CF individuals eluted in the void volume on Sepharose 4B, suggesting that CF tracheobronchial mucins were not inherently smaller than non-CF mucins.

Structure of the carbohydrate portion of the sialoglycopeptide isolated from the skin of the fish Mastacembalus armatus

The sialoglycopeptide (PSGP-A), isolated from the degelled skin of the fish Mastacembalus armatus, contained sialic acids (NANA and NGNA, 13.5%), GlcNAc (19.3%), Gal (14.7%), Man (12.6%), Fux (2.5%), and amino acids of which Asp, Ser, Thr, and Pro preponderated. Affinity chromatography showed that the two sialic acids were integral parts of the same glycopeptide molecules. The carbohydrate portion was linked through GlcNAc to Asn of the peptide core. Methylation analysis of PSGP-A and its degradation products and periodate-oxidation studies indicated a structure for the carbohydrate portion of PSGP-A which resembled those of other sialoglycopeptides having similar carbohydrate compositions.

Erythro- and lymphoagglutinins of Phaseolus acutifolius

A potent lymphoagglutinin which had low affinity for red cells or fetuin and another lectin which reacted strongly with red cells and fetuin but was a poor agglutinin for lymphocytes were isolated from seeds of Phaseolus acutifolius. A number of other lectin components with intermediate activity towards these cells was also isolated. All the lectins had very similar amino acid and carbohydrate composition, sedimentation patterns, partial specific volume and molecular weight values of about 116 600 and were thus smaller than the related Phaseolus vulgaris lectins (M r = 119 000). The lectins contained four subunits with only minor size and charge differences between the lympho- and erythroagglutinating subunits and their electrophoretic mobility in SDS gel electrophoresis was anomalously high. The existence of lympho- and erythroagglutinating subunits in two members of the genus Phaseolus supports their close morphological similarity.

Reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer.

Species-specific reaggregation of cells from the marine sponge Microciona prolifera is mediated by a proteoglycan-like aggregation factor (MAF) of Mr = 2 X 10(7) which has two functional domains, a cell binding domain and an aggregation factor interaction domain. After extensive trypsin digestion, over 60% of the MAF mass was converted into a glycopeptide fragment of Mr = 10,000 (T-10) which is therefore a representative part of the major portion, but not of the entire MAF molecule. The T-10 fragment has a similar amino acid and carbohydrate composition as the intact MAF and displays species-specific binding. Although T-10 also inhibited MAF association with homotypic cells, its apparent affinity is 3 X 10(6) M-1, i.e. 13,000 times lower than that of native MAF. Reconstitution of binding affinity in the same order of magnitude as native MAF (Ka = 10(10) M-1) was obtained by cross-linking the glycopeptide fragment into polymers of the approximate size of MAF (Mr greater than 1.5 X 10(7) using diepoxybutane and glutaraldehyde, or periodate oxidation and glutaraldehyde. The apparent association constants of intermediate polymers with Mr = 1 X 10(5), 6 X 10(5), 9 X 10(5), 2 X 10(6) and above 1.5 X 10(7) increased proportionally to their size and were in line with association constants of MAF degradation fragments. Since the binding affinity of the T-10 glycopeptide fragment could be reconstituted by cross-linking, and since this fragment accounts for over 60% of MAF, we propose that the specificity and high affinity of the MAF-cell association is based on a highly polyvalent interaction of low affinity cell-binding sites. Such a polyvalency of the cell binding domain is advantageous for efficient cell-cell interactions and thus differs from most known interaction molecules and receptors characterized.

Glycoprotein-bound large carbohydrates of early embryonic cells: structural characteristic of the glycan isolated from F9 embryonal carcinoma cells.

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.