Thiophilic adsorption chromatography: purification of Equ c2 and Equ c3, two horse allergens from horse sweat

Purification of two allergens from horse (Equus caballus) sweat, Equ c2 and Equ c3, by means of salt-promoted chromatography on a “thiophilic” (T-gel) adsorbent is described. Immobilization of these proteins was found to be dependent on the presence of water-structure-forming salts where the ammonium sulphate concentration in the equilibration buffer was 2 M. Equ c2 showed higher affinity towards the thiophilic matrix than Equ c3. Their molecular mass (M_r) values established by SDS–polyacrylamide gel electrophoresis were for Equ c2 ≈17 000 and for Equ c3 ≈16 000, and both proteins showed a low isoelectric point of ≈3.8. Their allergenic properties were also investigated using sera from horse-sensitized patients, where it was demonstrated that these proteins exhibited an IgE antibody binding capacity. In this report we show the broad potential applications of thiophilic adsorption chromatography for the efficient purification of allergens.     

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.     

Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Le x and relevant compounds

From the beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (1) prepared by the transglycosylation of beta-galactosidase from Bacillus circulans, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (9) and alpha-D-Neu5Ac-(2-->6)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (10) were effectively synthesized with an equimolar ratio of CMP-Neu5Ac by recombinant rat alpha-(2-->3)-N-sialyltransferase and rat liver alpha-(2-->6)-N-sialyltransferase, respectively. The former enzyme also transferred effectively the Neu5Ac residue from CMP-Neu5Ac to the location of OH-3 in the non-reducing terminal of beta-D-Gal-(1-->4)-beta-D-Gal-OC6H4NO2-p or beta-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p, while the latter enzyme did not. In the case of equimolar ratio of GDP-Fuc/acceptor, 1 and 9 were further fucosylated quantitatively to form beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (14) and alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (13) by recombinant human alpha-(1-->3)-fucosyltransferase VII, respectively.     

Two new alkaloidal taxoids from the needles of Taxus canadensis

Two new taxanes with a dimethylamino group on the C-5 side chain were identified for the first time in the needles of the Canadian yew, Taxus canadensis. Their structures were characterized as 7beta,10beta,13alpha-triacetoxy-5alpha-(3'-dimethylamino-3'-phenylpropanoyl)oxy-2alpha-hydroxy-2(3-->20)abeotaxa-4(20),11-dien-9-one (1) and 2alpha,10beta-diacetoxy-9alpha-hydroxy-5alpha-(3'-dimethylamino-3'-phenylpropanoyl)oxy-3,11-cyclotax-4(20)-en-13-one (2).     

An enzymatically produced novel cyclomaltopentaose cyclized from amylose by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans, produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. The other minor product was cyclomaltohexaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one alpha-(1-->6)-linkage. ICG5 was digested by alpha-amylase derived from Aspergillus oryzae, cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus, and maltogenic alpha-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans, and maltogenic alpha-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an alpha-(1-->6)-linkage in their molecules.     

Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase

Cellobiose was tested as acceptor in the reaction catalyzed by alternansucrase (EC 2.4.1.140) from Leuconostoc mesenteroides NRRL B-23192. The oligosaccharides synthesized were compared to those obtained with dextransucrase from L. mesenteroides NRRL B-512F. With alternansucrase and dextransucrase, overall oligosaccharide synthesis yield reached 30 and 14%, respectively, showing that alternansucrase is more efficient than dextransucrase for cellobiose glucosylation. Interestingly, alternansucrase produced a series of oligosaccharides from cellobiose. Their structure was determined by mass spectrometry and [13C-1H] NMR spectroscopy. Two trisaccharides are first produced: alpha-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-D-glucopyranose (compound A) and alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->4)-D-glucopyranose (compound B). Then, compound B can in turn be glucosylated leading to the synthesis of a tetrasaccharide with an additional alpha-(1-->6) linkage at the non-reducing end (compound D). The presence of the alpha-(1-->3) linkage occurred only in the pentasaccharides (compounds C1 and C2) formed from tetrasaccharide D. Compounds B, C1, C2 and D were never described before. They were produced efficiently only by alternansucrase. Their presence emphasizes the difference existing in the acceptor reaction selectivity of the various glucansucrases.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and methylation analysis

Two galactofuranomannans, Ths-4 and Ths-5, were isolated from the lichen, Thamnolia vermicularis var. subuliformis, using ethanol fractionation and anion-exchange and size-exclusion chromatography. The average molecular weights of Ths-4 and Ths-5 were estimated to be 19 and 200 kDa, respectively. Structural characterisation of Ths-4, Ths-5 and their partially hydrolysed derivatives was performed by methanolysis and methylation analysis. The intact and partially hydrolysed Ths-4 was further analysed using NMR spectroscopy (1D, COSY, NOESY, TOCSY, HSQC and HMBC). According to the data obtained, the heteroglycans Ths-4 and Ths-5 have similar structures, but have large differences in molecular weight. The structure is composed of 3-O-linked and 5-O-linked galactofuranosyl chains linked to a mannan core. The mannan core consists of a main chain of alpha-(1-->6)-linked mannopyranosyl residues, substituted at O-2 with either a single alpha-mannopyranosyl unit or an alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown.     

Secondary metabolites from Senecio burtonii (Compositae)

A cacalolide derivative named 4alpha-[2'-hydroxymethylacryloxy]-1beta-hydroxy-14-(5-->6) abeo eremophilan-12,8-olide and a shikimic acid derivative named (3'E)-(1alpha)-3-hydroxymethyl-4beta,5alpha-dimethoxycyclohex-2-enyloctadec-3'-enoate along with three known compounds, octacosan-1-ol, 3beta-hydroxyolean-12-en-28-oic acid and 3beta-acetoxyolean-12-en-28-oic acid were isolated from Senecio burtonii. Their structures and relative configurations were established on the basis of spectroscopic analysis.     

Effect of dietary cyclic nigerosylnigerose on intestinal immune functions in mice

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.     

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6 microg/ml were much lower than the cytotoxic concentration 50% (CC50) values (1000 microg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60-100 microg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160 kDa, contained a backbone of alpha-(1-->3)-linked D-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of beta-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of alpha-(1-->3)-linked D-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.     

Lactones of disialyl lactose: characterisation by NMR and mass spectra

The lactonisation of alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose) was investigated. (1)H and (13)C NMR chemical shifts of disialyl lactose and alpha-Neup5Ac-(2-->8, 1-->9)-alpha-Neup5Ac-(2-->3, 1-->2)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose-dilactone) were assigned based on 1D and 2D NMR results, including edited HSQC, HSQC-TOSCY and HMBC. The time course of lactonisation was followed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with electrospray ionisation (ESI) mass spectrometry (MS) detection. The rate of lactonisation between alpha-(8)Neu5Ac and alpha-(3)Neu5Ac residues (lactonisation at the alpha-(2-->8) linkage) was faster than that of lactonisation between alpha-(3)Neu5Ac and Gal residues (lactonisation at the alpha-(2-->3) linkage). The mass spectra of disialyl lactose, its lactones, alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac (alpha-(2-->8) disialic acid) and alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc-lactone (3'-sialyllactose-lactone) showed that the alpha-(2-->8) linkage between Neu5Ac residues is difficult to cleave in the ESI-MS, compared with the alpha-(2-->3) linkage between Neu5Ac and Gal residues.     

Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo [-->6]-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->)) from alpha-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-alpha-glucosyltransferase (6GT) catalyzing the a-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce alpha-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing alpha-1,3-, alpha-1,4-, and alpha,beta-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed alpha-1,3-transglycosylation, alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS was produced from alpha-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS-->alpha-isomaltosyl-->MOS (by 6GT), 2) alpha-isomaltosyl-MOS-->alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS (by IMT), and 3) alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS-->CTS + MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45 degrees C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40 degrees C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50 degrees C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40 degrees C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.     

Purification, characterization, and gene cloning of a novel maltosyltransferase from an Arthrobacter globiformis strain that produces an alternating alpha-1,4- and alpha-1,6-cyclic tetrasaccharide from starch

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50 degrees C and was stable from pH 5.0 to 9.0 and up to 30 degrees C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45 degrees C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of > or =3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose --> 6(4)-O-alpha-maltosyl-maltotetraose + maltose and (ii) 6(4)-O-alpha-maltosyl-maltotetraose --> CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular alpha-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to alpha-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.     

Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.     

Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgE-reactive epitopes

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 A resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a beta-barrel flanked by a C-terminal alpha-helix. The space between the two beta-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.     

Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of fructo-oligosaccharides in vivo

A fructosyltransferase that transfers the terminal (2 --> 1)-beta-linked D-fructosyl group of fructo-oligosaccharides (1(F)(1-beta-D-fructofuranosyl)(n) sucrose, n >/= 1) to HO-6 of the glucosyl residue and HO-1 of the fructosyl residue of similar saccharides (1(F)(1-beta-D-fructofuranosyl)(m) sucrose, m >/= 0) has been purified from an extract of the bulbs of onion (Allium cepa). Successive column chromatography using DEAE-Sepharose CL-6B, Toyopearl HW65, Toyopearl HW55, DEAE-Sepharose CL-6B (2nd time), Sephadex G-100, Concanavalin A Sepharose, and Toyopearl HW-65 (2nd time) were applied for protein purification. The general properties of the enzyme, were as follows: molecular masses of 66 kDa (gel filtration chromatography), and of 52 kDa and 25 kDa (SDS-PAGE); optimum pH of c. 5.68, stable at 20-40 degrees C for 15 min; stable in a range of pH 5.30-6.31 at 30 degrees C for 30 min, inhibited by Hg(2+), Ag(+), p-chloromercuribenzoic acid (p-CMB) and sodium dodecyl sulfate (SDS), activated by sodium deoxycholate, Triton X-100 and Tween-80. The amino acid sequence of the N-terminus moiety of the 52-kDa polypeptide was ADNEFPWTNDMLAWQRCGFHFRTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYXG and the amino acid sequence from the N-terminus of the 25-kDa polypeptide was ADVGYXCSTSGGAATRGTLGPFGLL VLANQDLTENTATYFYVSKGTDGALRTHFCQDET. The enzyme tentatively classified as fructan: fructan 6(G)-fructosyltransferase (6G-FFT). The enzyme is proposed to play an important role in the synthesis of inulin and inulinneo-series fructo-oligosaccharides in onion bulbs.     

Purification and characterization of a novel alpha-glucuronidase from Aspergillus niger specific for O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid

A new alpha-glucuronidase that specifically hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-alpha-D-glucosyluronic acid alpha-D-glucoside, O-alpha-D-glucosyluronic acid beta-D-glucosiduronic acid, O-alpha-D-glucosyluronic acid-(1-->2)-beta-D-fructosiduronic acid, p-nitrophenyl-O-alpha-D-glucosiduronic acid, methyl-O-alpha-D-glucosiduronic acid, or 6-O-alpha-(4-O-alpha-D-glucosyluronic acid)-D-glucosyl-beta-cyclodextrine. Furthermore, it showed no activity on alpha-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-alpha-(1-->2)-xylooligosaccharides, derived from xylan, a supposed substrate of alpha-glucuronidases.The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS-PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0-3.5 and at 40 degrees C. It was stable in pH 2.0-4.5 and below 30 degrees C. It hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid to produce alpha- and beta-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)