cDNA cloning of β-tubulin gene and organization of tubulin genes in Vigna radiata (mung bean) genome

We isolated an almost full-length cDNA clone containing -tubulin gene from a partial cDNA library of mung bean using chicken cDNA as probe. Cross-hybridization with chicken -tubulin cDNA and positive hybridization-selection and translation of mung bean mRNA established that this clone contains -tubulin sequences. We studied the organization of tubulin genes in mung bean. In this plant tubulin genes are organized in tandem repeats of alternating - and -tubulin genes. The 5.6 kb basis repeat unit which contains both - and -tubulin genes is repeated twenty times per haploid genome.Abbreviations SDS sodium dodecyl sulphate - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA, pH 7.4     

The contribution of surface residues to membrane binding and ligand transfer by the α-tocopherol transfer protein (α-TTP)

Previous work has shown that the α-tocopherol transfer protein (α-TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which α-TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of α-TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of α-TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired α-TTP-assisted secretion of α-tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.     

Exploring the Ultrastructural Localization and Biosynthesis of β(1,4)-Galactan in Pinus radiata Compression Wood

Softwood species such as pines react to gravitropic stimuli by producing compression wood, which unlike normal wood contains significant amounts of β(1,4)-galactan. Currently, little is known regarding the biosynthesis or physiological function of this polymer or the regulation of its deposition. The subcellular location of β(1,4)-galactan in developing tracheids was investigated in Pinus radiata D. Don using anti-β(1,4)-galactan antibodies to gain insight into its possible physiological role in compression wood. β(1,4)-Galactan was prominent and evenly distributed throughout the S2 layer of developing tracheid cell walls in P. radiata compression wood. In contrast, β(1,4)-galactan was not detected in normal wood. Greatly reduced antibody labeling was observed in fully lignified compression wood tracheids, implying that lignification results in masking of the epitope. To begin to understand the biosynthesis of galactan and its regulation, an assay was developed to monitor the enzyme that elongates the β(1,4)-galactan backbone in pine. A β(1,4)-galactosyltransferase (GalT) activity capable of extending 2-aminopyridine-labeled galacto-oligosaccharides was found to be associated with microsomes. Digestion of the enzymatic products using a β(1,4)-specific endogalactanase confirmed the production of β(1,4)-galactan by this enzyme. This GalT activity was substantially higher in compression wood relative to normal wood. Characterization of the identified pine GalT enzyme activity revealed pH and temperature optima of 7.0 and 20°C, respectively. The β(1,4)-galactan produced by the pine GalT had a higher degree of polymerization than most pectic galactans found in angiosperms. This observation is consistent with the high degree of polymerization of the naturally occurring β(1,4)-galactan in pine.The ability to respond to gravitropic stimuli is important for the survival of most terrestrial plants. Arborescent angiosperm and gymnosperm species generate wood with modified properties, called reaction wood, in response to gravitropic stimuli (Timell, 1969, 1986; Du and Yamamoto, 2007). The formation of reaction wood enables the return of bent stems to a vertical orientation. Interestingly, the location and type of the reaction wood deposited in woody gymnosperms and angiosperms generally differs significantly. Gymnosperms respond to gravitropic stimuli by compression wood formation on the underside of leaning stems (Timell, 1986), and arboreal angiosperms generate reaction wood primarily in the form of tension wood on the upper side of inclined stems (Timell, 1969).Compression wood in conifers differs significantly from normal wood in its anatomical, chemical, and physical properties. Typical anatomical features of severe compression wood are short, rounded, and thick-walled tracheids with a prominent band of lignin in the outer S2 layer of the cell wall as well as spiral checks and the absence of an S3 layer (Timell, 1986). Biochemically, compression wood is characterized by high levels of lignin, rich in condensed p-hydroxyphenyl units, as well as reduced cellulose and galactoglucomannan relative to normal wood (Timell, 1986; Nanayakkara et al., 2005; Yeh et al., 2006). Most striking, though, is that β(1,4)-galactan can constitute more than 10% (w/w) of the cell wall material in severe compression wood but is virtually absent in normal wood (Nanayakkara et al., 2005; Yeh et al., 2006). Recent work suggests that β(1,4)-galactan biosynthesis represents an early step in compression wood formation and confirms that its presence is diagnostic for this wood type (Altaner et al., 2007). However, the molecular signal cascades in conifers that lead to the deposition of β(1,4)-galactan are currently not well understood.Immunological studies in conifer species using the monoclonal anti-β(1,4)-galactan LM5 antibody (Jones et al., 1997) indicate that β(1,4)-galactan in compression wood is located in the S1 and outer S2 layers of mature tracheids but is virtually absent from the primary cell walls (Schmitt et al., 2006; Altaner et al., 2007; Möller and Singh, 2007). Instead of β(1,4)-galactan, most conifers contain small amounts of arabinogalactan, a polysaccharide characterized by a highly branched β(1,3)-galactan backbone (Vikkula et al., 1997; Willför et al., 2002; Laine et al., 2004) in their primary cell walls. The ultrastructural distribution of β(1,4)-galactan in compression wood appears to be largely consistent with highly lignified cell wall layers (Möller and Singh, 2007), which might explain the involvement of β(1,4)-galactan in the formation of lignin-carbohydrate complexes (Mukoyoshi et al., 1981; Minor, 1982; Timell, 1986; Laine et al., 2004).The investigation of β(1,4)-galactan structure in preparations from Pinus sylvestris (Laine et al., 2004) and Pinus radiata (Nanayakkara 2007) revealed a linear polymer. In Pinus densiflora Siebold & Zucc., β(1,4)-galactan was found to be slightly branched at positions C2, C3, and C6 (Mukoyoshi et al., 1981). β(1,4)-Galactan in conifers display a high degree of polymerization (DP), which was originally estimated to be in the range of 200 to 300 (Timell, 1986). More recent studies with P. radiata compression wood found the native polysaccharide to have a DP of approximately 380 (Nanayakkara 2007).β(1,4)-Galactan is a very good biochemical marker for compression wood (Altaner et al., 2007), but its physiological role is currently not well understood. Various functions for β(1,4)-galactan in compression wood have been proposed, such as strengthening of the secondary cell wall, absorption of mechanical stresses, and generation of compressive forces (Möller and Singh, 2007). Furthermore, β(1,4)-galactan is also found in tension wood, with a proposed role in cross-linking cellulose microfibrils (Arend, 2008). However, all of those hypotheses on the molecular function of β(1,4)-galactan in reaction wood await experimental verification.Despite substantial efforts to characterize the biosynthesis of this polymer, β(1,4)-galactan biosynthetic enzymes and their corresponding genes are currently unknown (Peugnet et al., 2001; Geshi et al., 2002, 2004; Abdel-Massih et al., 2003; Kato et al., 2003; Ishii et al., 2004; Konishi et al., 2004, 2007; Gorshkova and Morvan, 2006). However, based on other cell wall polysaccharide biosynthetic enzymes, it is likely that the enzymes involved in the biosynthesis of β(1,4)-galactan are either Golgi localized or pass through the Golgi in transit to the apoplastic space (Reyes and Orellana, 2008).To better understand β(1,4)-galactan synthesis in compression wood formation, we sampled both normal wood and severe compression wood from two 6-year-old P. radiata trees, which displayed stark differences in lignin and carbohydrate content and composition. Using these wood samples, new insights into the subcellular localization of β(1,4)-galactan in pine were generated using confocal laser fluorescence microscopy and transmission electron microscopy. An enzyme assay was developed, based on 2-aminopyridine (2AP)-labeled galacto-oligosaccharides as acceptor molecules, which we used to identify and partially purify a robust, microsome-associated, UDP-Gal-dependent β(1,4)-galactosyltransferase (GalT) activity in compression wood that was virtually undetectable in normal wood. Assays of the partially purified GalT revealed that this enzyme has some properties similar to those of previously characterized pectic GalTs, but a marked difference was observed in the size distribution of the enzymatic products.     

Identification of elongating β-1,4-galactosyltransferase activity in mung bean (<Emphasis Type="Italic">Vigna radiata</Emphasis>) hypocotyls using 2-aminobenzaminated 1,4-linked β-<Emphasis Type="SmallCaps">d</Emphasis>-galactooligosaccharides as acceptor substrates

Galactosyltransferase (GalT) activity that results in the transfer of galactose (Gal) from UDP-Gal to exogenous (14)--galactooligosaccharides labeled with 2-aminobenzamide (2AB) at their reducing ends was identified in a particulate preparation obtained from 2-day-old mung bean (Vigna radiata L. Wilezek) hypocotyls. The enzymes responsible were shown, by high-performance anion-exchange chromatography and normal-phase liquid chromatography–electrospray ionization mass spectrometry, to transfer up to eight Gals to the non-reducing end of 2AB-labeled galactooligosaccharide. Using ¹H nuclear magnetic resonance spectroscopy, and -galactosidase and endo--(14)-galactanase treatments of the enzymatically formed 2AB-labeled galactooligosaccharides, the newly incorporated Gal residues were shown to be -(14) linked. Time-course studies indicated that at least two different types of GalT isoform are involved in the elongation of the acceptor substrates. 2AB-labeled galactoheptaose was the most effective acceptor substrate analyzed, although galactooligosaccharides with a degree of polymerization between 4 and 6 were also acceptor substrates. 2AB-labeled penta- and heptasaccharides (RG₅ and RG₇) generated from rhamnogalacturonan I (RG-I) were not acceptor substrates, suggesting that the GalTs were not capable of adding Gal residues directly to the RG-I backbone. Maximum GalT activity was obtained at pH 6.5 and 20°C in the presence of 25 mM Mn²⁺ and 0.75% (w/v) Triton X-100. The enzyme had an apparent K_m of 20 M for 2AB-labeled galactoheptaose and 32 M for UDP-Gal. The characteristics of the enzyme in mung bean microsomal membranes and the usefulness of fluorogenic 2AB-labeled galactooligosaccharides for the assay of GalT are discussed.Abbreviations 2AB 2-Aminobenzamide - DP Degree of polymerization - Gal Galactose - GalT Galactosyltransferase - HPAEC High-performance anion-exchange chromatography - LC–ESI–MS Liquid chromatography–electrospray ionization mass spectrometry - RG-I Rhamnogalacturonan I - SEC Size exclusion chromatography     

Hydrolysis of raffinose and stachyose in cowpea (Vigna unguiculata) flour,using α-galactosidase fromAspergillus niger

Filamentous fungi, isolated from the enriched surface of garden soil, were screened for -galactosidase production after growing on wheat-bran/carbohydrate substrate. One isolate,Aspergillus niger, had the highest enzyme activity (5.1×10^–2 units/mg protein) at pH 5.0 and 50°C. Treatment of cowpea flour with the crude enzyme reduced the raffinose and stachyose content by 95% and 82% respectively. This technique could therefore be useful in controlling the flatulence activity of cowpeas.     

Alleviation of drought stress in faba bean (Vicia faba L.) by exogenous application of β-aminobutyric acid (BABA)

Physiology and Molecular Biology of Plants - Drought stress is one of the most prevalent environmental factors limiting faba bean (Vicia faba L.) crop productivity. β-aminobutyric acid (BABA)...     

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.     

Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.     

Purification and characterization of a soluble β-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble β-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182–191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble β-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the β-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this β-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.     

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcβ1,4GlcNAcβ. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in β1,4-linkage to GlcNAc in vivo and in vitro. We now show that β4GalNAc-T3 and β4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to β1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galβ1,3(GalNAcβ1,4GlcNAcβ1,6)GalNAcαSer/Thr. The β1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcβ1,4GlcNAcβ (LacdiNAc)-containing structures on specific glycoproteins.     

Development and validation of competition binding assays for affinity to the extracellular matrix receptors, α(v)β(3) and α(IIb)β(3) integrin

The RGD (Arg-Gly-Asp) binding integrins α(v)β(3) and α(IIb)β(3) are integral components of various pathological and physiological processes, including tumor angiogenesis, osteoclast function, and thrombus formation. Because of this, there is interest in identifying novel compounds and proteins binding to these receptors as well as investigating the mechanism of these interactions. In this article, we describe the development and validation of competition binding assays for determining the affinity of test compounds to α(v)β(3) and α(IIb)β(3) integrin. Assays were successfully developed for each receptor, and the affinity of known compounds was comparable to published results. However, the inability of binding between α(IIb)β(3) integrin and the labeled echistatin protein ligand to reach equilibrium resulted in an assay that did not meet the assumptions of the competition binding model. Nevertheless, there was good agreement between this assay and known literature values, and intra- and interassay variability was acceptable. Binding by conformation-specific antibodies provided evidence that solid-phase bound α(IIb)β(3) receptor was in an activated conformation. This study also demonstrated that current models and methods for determining receptor affinity are simplistic and fail to account for common receptor-ligand interactions such as nondissociable interactions and varying receptor activation states.     

Effect of α(2–6)-linked sialic acid and α(1–3)-linked fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA)

The effects of branching and substitution of branches by sialic acid and fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA) were examined. Asialo bi-, tri-and tetra-antennary glycans were all retarded but to different extents on a long column of L-PHA-agarose. Asialo tri- and tetra-antennary glycans containing the pentasaccharide unit Gal1-4GlcNAc1-2[Gal1-4GlcNAc1-6]Man were strongly retarded, whereas asialo bi- and tri-antennary glycans lacking the Gal1-4GlcNAc1-6 branch were only weakly retarded. In all instances the interaction with the lectin was completely abolished when either (2–6)-linkedN-acetylneuraminic acid or (1–3)-linked fucose was present at the galactose orN-acetylglucosamine residue of the Gal1-4GlcNAc1-6Man1-6 branch, respectively. The same substitutions on the Gal1-4GlcNAc1-6Man1-6 branch decreased but did not abolish the affinity of the lectin for the glycans. The presence of NeuAc2-6 and Fuc1-3 on the other two branches did not interfere with the binding of the glycans to L-PHA. Furthermore, it appeared that the presence of the Man1-4GlcNAc unit is requried for interaction with the lectin. In order to obtain reliable information on the relative occurrence of tri- and tetra-antennary glycopeptides, this study shows that it is essential to desialylate and to defucosylate the glycans prior to application to L-PHA-agarose.Abbreviations L-PHA leukoagglutinating phytohemagglutinin - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - GP glycopeptide - OS oligosaccharide - HPLC high-performance liquid chromatography - FNR fraction not retarded - FR fraction retarded suffixes MS, BS and TS indicate mono-, bi- and trisialyl derivatives respectively; suffix MF indicates monofucosyl derivatives.structures of the substratesOS2, OS3, OS3, OS4, GP2, GP3, GP4, GP4-MF, OS2(3) andOS2(-) are presented in Fig. 2     

Jack bean α-mannosidase (Jbα-man): tolerance to alkali, chelating and reducing agents and energetics of catalysis and inhibition

Investigations of the catalytic and structural transitions of jack bean α-mannosidase (Jbα-man) are described in the present paper. The enzyme was maximally stable at pH 5.0; however, when incubated in the pH range of 11.0-12.0, showed 1.3 times higher activity and also stability for longer time. The free amino group at or near the active site was probably involved in the stability and activation mechanism. The active site is constituted by the association of two unidentical subunits connected by disulfide linkages. The metalloenzyme has Zn²⁺ ions tightly bound and chelation reduces the thermal stability of the protein. Energetics of catalysis and thermodynamics of inhibition of the enzyme were also carried out.     

Production of rare ginsenosides (compound Mc, compound Y and aglycon protopanaxadiol) by β-glucosidase from Dictyoglomus turgidum that hydrolyzes β-linked, but not α-linked, sugars in ginsenosides

Optimal hydrolytic activity of β-glucosidase from Dictyoglomus turgidum for the ginsenoside Rd was at pH 5.5 and 80?°C, with a half-life of ~11?h. The enzyme hydrolysed β-linked, but not α-linked, sugar moieties of ginsenosides. It produced the rare ginsenosides, aglycon protopanaxadiol (APPD), compounds Y, and Mc, via three unique transformation pathways: Rb(1)?→?Rd?→?F(2)?→?compound K?→?APPD, Rb(2)?→?compound Y, and Rc?→?compound Mc. The enzyme converted 0.5?mM Rb(2) and 0.5?mM Rc to 0.5?mM compound Y and 0.5?mM compound Mc after 3?h, respectively, with molar conversion yields of 100?%.     

The homology of the major storage protein of jack bean (Canavalia ensiformis) to pea vicilin and its separation from α-mannosidase

The major storage protein of jackbean (Canavalia ensiformis) has been purified by a protocol involving ammonium-sulphate precipitation, gel filtration and ion-exchange chromatography. The protein was shown by partial amino-acid-sequence data to be homologous to vicilin, a major storage protein of pea (Pisum sativum), and is thus a member of the family of legume 7S proteins exemplified by pea vicilin. This protein is thus referred to as jack-bean vicilin rather than canavalin or precanavalin as previously used. Other properties of the jack-bean vicilin (e.g. subunit relative molecular mass (M_r) and structure, resistance to proteolysis) show similarity to phaseolin, the major 7S storage protein ofPhaseolus vulgaris. Jack-bean vicilin contained no detectable -mannosidase activity, either as isolated from mature or germinating seeds, or after proteolytic treatment. -Mannosidase was also purified from jack beans, and was shown to have a subunit M_r of approx. 120,000; it was separated completely from jack-bean vicilin by a similar protocol to that used for purifying the latter. The -mannosidase was proteolytically cleaved after seed germination, but did not give polypeptides of the same M_r as jackbean vicilin. It was concluded that -mannosidase and jack-bean vicilin are not related proteins.Abbreviations DE diethylaminoethyl - M relative molecular mass - SDS sodium dodecyl sulphate - PAGE polyacrylamide-gel electrophoresis     

Purification and Characterization of β-D-Glucosidase (β-D-Fucosidase) from Bifidobacterium breve clb Acclimated to Cellobiose

The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants.     

Molecular Basis for Protein-specific Transfer of N-Acetylgalactosamine to N-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

Two closely related β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, are thought to account for the protein-specific addition of β1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein hormone luteinizing hormone and carbonic anhydrase-6 (CA6). We have utilized soluble, secreted forms of β4GalNAc-T3 and β4GalNAc-T4 to define the basis for protein-specific GalNAc transfer in vitro to chimeric substrates consisting of Gaussia luciferase followed by a glycoprotein substrate. Transfer of GalNAc by β4GalNAc-T3 and β4GalNAc-T4 to terminal GlcNAc is divalent cation-dependent. Transfer of GalNAc to glycoprotein acceptors that contain a peptide recognition determinant is maximal between 0.5 and 1.0 mm MnCl(2); however, transfer is increasingly inhibited by concentrations of MnCl(2) above 1 mm and by anion concentrations above 15 mm. In contrast, transfer of GalNAc to the simple sugar acceptor N-acetylglucosamine-β-p-nitrophenol (GlcNAcβ-pNP) is not inhibited by concentrations of MnCl(2) or anions that would inhibit transfer to glycoprotein acceptors by >90%. This finding indicates that interaction with the peptide recognition determinant in the substrate is sensitive to the anion concentration. β4GalNAc-T3 and β4GalNAc-T4 have similar but distinct specificities, resulting in a 42-fold difference in the IC(50) for transfer of GalNAc to chimeric glycoprotein substrates by agalacto human chorionic gonadotropin, comprising 29 nm for β4GalNAc-T3 and 1.2 μm for β4GalNAc-T4. Our in vitro analysis indicates that enzymatic recognition of the peptide determinant and the oligosaccharide acceptor are independent events.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

Production and properties of carboxymethylcellulase (endo-1,4-β-glucanase) fromCurvularia lunata

An extracellular carboxymethylcellulase (endo-1,4--glucanase) fromCurvularia lunata, grown at 30°C with an initial pH of 6.0, had optimal activity at pH 4.8 and 50°C. The enzyme was unstable above 50°C. The enzyme had aK _m for carboxymethylcellulose of 0.97 g/l and aV _max of 5.4 IU/ml.