Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 -glycosidase (Tca -glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a M _r of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca -glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg^–1 protein.     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄     

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

The crystal-forming proteins (-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (M _r of ca. 145,000–130,000) contain an N-terminal domain (M _r of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (M _r of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with M _r of 15,000–35,000.The N-terminal domain from serotype V -endotoxin is active when introduced into the hemocoel of Galleria mellonella larvae. Hence, it correponds to the true toxin normally formed by larva proteases action on the crystalforming protein (protoxin). Some differences were found in the properties of the N-terminal domains isolated from the crystal-forming proteins of III, V and IX serotypes, e.g., in their solubility, digestion by subtilisin, molecular weights and the distribution of methionine residues along the polypeptide chains.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacryl amide gel electrophoresis - CFP crystal-forming protein - DNS 5-dimethylamino-1-naphthalene-sulphonyl     

Multiple molecular forms of β-amylase in seeds and vegetative tissues of barley

The molecular forms of -amylase present in developing, mature, germinating and malted grains of barley (Hordeum vulgare L.), and in vegetative tissues, have been studied using Western-blot analyses and isoelectric focusing of isoenzymes. Five isoforms with different relative molecular masses (M_rs) could be recognised. The major isoform present in the mature grain, called isoform B, had an M_r of about 60 000. This was converted on malting or germination to two lower-M_r forms called C and D. Previous work (R. Lundgard and B. Svensson, 1986, Carlsberg Res. Commun. 51, 487–491) has shown that these result from partial proteolysis of isoform B. Isoenzyme analyses showed complex patterns of bands, with pIs between about 5.0 and 6.0. Two allelic types were present in the eight lines. A number of new bands with a range of pIs appeared during germination and malting.An isoform with the same M_r as D and a minor low-M_r isoform (E) were present in young developing whole caryopses (8–12 d after anthesis), but not in older developing endosperms (14–21 d after anthesis). Isoenzyme analyses also showed different patterns of bands in these two tissues, while hybrid-dot analyses indicated the presence of separate populations of mRNAs. It is suggested that the early endosperm isoforms (D and E) are green -amylases present in the pericarp and-or testa of the young caryopses.Roots but not shoots or leaves also contained an isoform with the same M_r as D, although the pattern of isoenzymes differed from that present in the seed tissues.The fifth isoform, A, was a diffuse high-M_r form present in small amounts in all seed and vegetative tissues, and may correspond to a constitutively expressed form.These multiple molecular forms of -amylase are discussed in relation to the recent report that -amylase is encoded by two structural loci, with a total copy number of two to three per haploid genome (Kreis et al, 1988, Genet. Res. Camb. 51, 13–16).Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

Is crassulacean acid metabolism activity in sympatric species of hemi-epiphytic stranglers such as Clusia related to carbon cycling as a photoprotective process?

A comparison of carbon isotope discrimination characteristics, crassulacean acid metabolism (CAM) activity and gas exchange together with concurrent analysis of photosystem II (PSII) chlorophyll fluorescence was conducted on leaves of sympatric species of Clusia from the restinga of Barra de Maricá, Brazil. The carbon isotope discrimination () and leaf-sap titratable acidity for leaves collected in the field indicated that the carbon metabolism of one species, C. lanceolata, was predominantly C3-like, and a second, C. fluminensis, constitutive CAM. When well-watered under glasshouse conditions C. lanceolata displayed a gas exchange pattern expected of a C3 plant, where values of instantaneous discrimination () rose from 13.5% shortly after dawn to 21.9 at midday, suggesting that all CO₂ uptake was mediated solely by ribulose 1,5-bisphosphate carboxylase (RUBISCO). C. fluminensis showed a gas exchange pattern which clearly exhibited all four phases of CAM. values during phase II ranged from –0.4 at dawn to 5.9 some 3 h later, indicating that C4 carboxylation dominated CO₂ uptake during the morning with an increasing contribution by RUBISCO, suggested by the 5 shift in at this time. The dominance of phosphoenol-pyruvate carboxylase (PEPc) activity was also found during phase IV, and extended throughout the dark period (phase I) in C. fluminensis, such that values of measured were negative (–5.0 to –0.4). This is the first time that negative values have been reported, close to those predicted theoretically for PEPc activity. The day-time uptake of CO₂ mediated by PEPc could lead to futile cycling through RUBISCO. In C. fluminensis organic acids were subjected to carbon turnover between PEPc and RUBISCO during phase II of CAM, serving perhaps to dissipate ATP and reductant at a time when excess photons are absorbed. Under low levels of photosynthetically active radiation (PAR) the two species displayed similar chlorophyll fluorescence characteristics, although for the CAM C. fluminensis a lower rate of decarboxylation of acids in the afternoon was reflected in changed quenching capacity. Under high PAR both species responded directly to changes in incident radiation, reflected by decreases in photon use efficiency (PSII) and the intrinsic photochemical efficiency (F _V/F _M), together with high and reversible quenching of excess light by the means of radiationless or thermal dissipation (q _N). Both species, with such markedly different carboxylation characteristics achieve similar rates of electron transport and maintain photosynthetic integrity. Under field conditions, however the severity of a prolonged dry season caused the CAM species to become deciduous, whereas the C3-like species remained healthy. This suggests that the widely expected advantages of CAM do not extend to tolerance of extreme environmental conditions, in contrast to the more C3-like of these sympatric species.     

Properties of α-galactosidase II2 from Vicia faba seeds

-Galactosidase II² (MW 43 390) from resting Vicia faba L. seeds had been shown to possess d-glucose/d-mannose-specific lectin activity. Inhibition studies with monosaccharides and an examination of the effects of heat and pH on the catalytic and lectin activities of the enzyme indicate that the enzyme substrate and the lectin haptens bind at different sites on the protein. d-Mannosebinding has been investigated by equilibrium dialysis and spectrophotometrically. Both methods yield K_a values of approx. 3·10³ M^-1 for the interaction and there would appear to be two mannosebinding sites per molecule of enzyme protein. The lectin properties of V. faba -galactosidase II² have been discussed in relation to both V. faba lectin (favin) and other legume -galactosidases.Abbreviations con A concanavalin A - CM-cellulose carboxymethyl cellulose - MW molecular weight - PNPG p-nitrophenyl -d-galactoside - SDS sodium dodecyl sulphate - PAGE polyacrylamide-gel electrophoresis     

Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum

Clostridium thermocellum, strain JW20 (ATCC 31449) when growing in cellulose produces a cellulolytic enzyme system, that at the early stage of the fermentation is largely bound to the substrate. As cellulose is consumed the bound enzyme is released as free enzyme to the culture fluid. The bound enzyme fraction extracted with distilled water from the cellulose contains two major components, a large complex (M_r100×10⁶) and a small complex M_r4.5×10⁶) which were separated by gel filtration and sucrose solved by affinity chromatography into a complex that binds to the column and into a non-bindable mixture of proteins. All four fractions have endo--glucanase activity but only the two bound complexes and the free bindable complex hydrolyze crystalline cellulose with cellobiose as the main product. These three complexes are qualitatively similar in that they each contain about 20 different polypeptides (M_r values from 45,000 to 200,000) of which about ten are major components. However, the relative amounts of some of the peptides in the complexes differ. At least four polypeptides of the complexes have endo--glucanase activity.Abbreviations CM cellulose, carboxymethyl cellulose - CMCase carboxymethyl cellulase cosidered endo--1,4-glucanase - SDS sodium dodecyl sulfate - YAS yellow affinity substance - YAS-cellulose yellow affinity substance-cellulose complex     

Symmetric interspecies hybrids of mouse and human hemoglobin: Molecular basis of their abnormal oxygen affinity

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ ₂ ^M ₂ ^H and ₂ ^H ₂ ^M (H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O₂ binding. The intrinsic O₂ affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid ₂ ^M ₂ ^H has high O₂ affinity (compared to either human or mouse Hb), while the interspecies hybrid ₂ ^H ₂ ^M exhibits a very low O₂ affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O₂ affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the _{1 1} (not the _{1 2}) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human ^S chains.     

Release of highly elicitor-active glucans by germinating zoospores of Phytophthora megasperma f. sp. glycinea

An in-vitro culture system allowing the simultaneous germination of cysts was used to study the early host-independent release of phytoalexin elicitors by Phytophthora megasperma f. sp. glycinea, a soybean pathogen. Significant elicitor activity could be detected in the culture medium as early as 2 h after germination of P.m. f. sp. glycinea, race 1, cysts. The phytoalexin elicitor was heat-stable and heterogeneous in size. The apparent molecular mass ranged from 3 to 80 kDa. Anion exchange and lectin-affinity chromatography followed by sugar analysis confirmed that the elicitor activity resided primarily in glucans. The time course of elicitor release could then be accurately monitored by means of a competitive radioligand-displacement assay using the -glucan elicitor-binding sites of soybean (Glycine max (L.) Merr.) membranes. Linkage-composition analysis of the glucan elicitors showed that they were primarily (1 3)-linked with (1 6)--branches, a composition similar to that of glucans obtained by heat release from mature mycelium but different from that of elicitors obtained by acid hydrolysis or from spontaneous autohydrolytic release by senescent cultures. The naturally released elicitors displayed a biological activity in soybean cotyledon bioassays higher than purified acid-hydrolysed glucan elicitor or than the hepta-(1 3, 1 6)--glucoside, the smallest known carbohydrate elicitor for soybean. The present results demonstrate that elicitor release from the pathogen and perception by the potential host can take place in this system as early as during germ-tube formation and independent of the presence of host-produced endoglucanases.Abbreviations EC₅₀ effector concentration necessary for halfmaximal response - GC-MS gas chromatography-mass spectrometry - HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-1-[hexagluco-syl]-deoxyglucitol - IC₅₀ inhibitor concentration necessary for half-maximal inhibition - P.m. f. sp. glycinea Phytophthora megasperma f. sp. glycinea - V₀ void volume Deceased on March 19, 1990This work was supported by the Deutsche Forschungsgemeinschaft (SFB 206). We thank Dr. H. Mayer, C. Warth and D. Borowiak (Max-Planck-Institut für Immunbiologie, Freiburg) for helpful discussions and experienced technical assistance (glucosyl-linkage analysis).     

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-β-(1→ 3),(1→ 4)-glucanase useful for the preparation of oligosaccharides from barley β-glucan

An endo--(13),(14)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo--(13),(14)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis, transformed with the recombinant plasmid pBLC771, produced an extracellular endo--(13),(14)-glucanase that was 130 times (7176 mU ml^–1) more active than that of the gene donor cells (55 mU ml^–1), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml^–1) more active than that of the gene donor cells. M _r of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble -glucan hydrolyzed by over-produced endo--(13),(14)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.     

High-affinity binding of fungal β-glucan elicitors to cell membranes of species of the plant family Fabaceae

Microsomal preparations of six species of the plant family Fabaceae were screened for high-affinity binding of branched (1 3), (1 6)--glucans. Oligoglucosides of this type are specific elicitors of phytoalexin accumulation in soybean (Glycine max L.), a member of this family. The species studied were alfalfa (Medicago sativa L.), broadbean (Vicia faba L.), chickpea (Cicer arietinum L.), french bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and white lupin (Lupinus albus L.). A ¹²⁵I-labeled 4-(2-aminophenyl)ethylamine conjugate of a (1 3), (1 6)--glucan fraction with an average degree of polymerization (DP) of 18, obtained from mycelial walls of Phytophthora sojae, was used as radioligand for initial screening. The structural complexity of this fraction allowed the identification of binding sites with affinities for isomeric structures other than the (1 3), (1 6) hepta--glucoside for which soybean binding sites display highest affinity. Radioligand competition experiments against unlabeled fungal -glucan resulted in the identification of high-affinity binding in alfalfa, bean, lupin, and pea. Half-maximal competition concentrations (IC₅₀) for fungal -glucan in these species were between 5 and 30 nM. Pseudoheterologous radioligand competition by unlabeled hepta--glucoside showed that for alfalfa, lupin and pea the IC₅₀ values for this structure (4 to 16 nM) were similar to those of soybean (7.7 nM). Bean microsomes, however, displayed an IC₅₀ significantly higher than soybean (68 nM) suggesting that the structural motif recognized by its binding sites is not identical to that of soybean or the other three species. Radioligand saturation assays with alfalfa, lupin and pea microsomes using an ₁₂₅I-labeled aminophenylethylamine hepta--glucoside conjugate gave dissociation constants (K_d) of 5.3, 3.7, and 1.8 nM, respectively. The affinity of these sites for hepta- glucoside was in the same range as that of soybean (K_d 1–3 nM), whereas the affinity of the binding sites of bean for the same ligand was significantly lower (K_d = 33 nM). Good correlation was found between the presence of high-affinity binding and the accumulation of isoflavonoid phytoalexins in roots of alfalfa, bean, chickpea and pea seedlings after exposure to fungal -glucan. Lupin displayed a strong wound-induced accumulation of prenylated isoflavones which was independent of the presence of -glucan, making it impossible to determine phytoalexin induction in response to elicitor. No specific binding or phytoalexin accumulation in response to glucans was observed in broadbean. This is the first report on the existence of possibly homologous elicitor-binding sites within a plant taxonomic family and may provide preliminary evidence for putative evolutionary relationships in pathogen perception mechanisms in plants.Abbreviations DP degree of polymerization - EC₅₀ concentration of elicitor necessary to obtain a half-maximal biological response - HG synthetic (1 3), (1 6)-hepta--glucoside phytoalexin elicitor - HG-APEA 1-[4-(2-aminophenyl)ethylamino-1-hexaglucosyl]deoxyglucitol - IC₅₀ ligand concentration necessary to obtain half-maximal displacement of radioligand in competition binding assays - K_d dissociation constant - OS branched (1 3), (1 6)--glucan obtained by hydrolysis of mycelial walls of Phytophthora sojae - OS-APEA 1-[4-(2-armnophenyl)ethylamino-1-oligoglucosyl]deoxyglucitol conjugate of OS This work was supported by the Comision Interministerial de Ciencia y Tecnologia grant BI091-0366 (E.G.C.), the Volkswagen-Stiftung (E.G.C. and J.E.), the Deutsche Forschungsgemeinschaft, SFB-369 (J.E.), the Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (J.E.), Fonds der Chemischen Industrie (J.E.) and the EU Human Capital and Mobility Program (J.E. and E.G.C.).     

Cloning and expression of the Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in Escherichia coli

A gene coding for the endo--1,3-1,4-glucanase of B. circulans ATCC21367 was cloned into Escherichia coli. The cloned enzyme hydrolyzed lichenan or barley -glucan to produce 3-O--cellobiosyl-d-glucose as a main product but was inactive with carboxymethyl cellulose, laminarin and xylan. The enzyme, M _r=28 kDa, remained within the cytoplasm of E. coli. A 771 bp open reading frame was in the 2 kb PstI fragment of the recombinant plasmid pLL200K. The deduced protein sequence consists of 257 amino acids and has a putative signal peptide of 26 amino acids. The amino acid sequence of the endo--1,3-1,4-glucanase showed 68 and 51% homology to previously reported endo--1,3-1,4-glucanases from Bacillus strain N-137 and B. brevis, respectively.     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM _r=52,000. During a 1.5-hr pulse-label with³⁵S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM _r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM _r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M _r=62,000), with the remainder retained intracellularly (M _r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM _r=56,000 that was processed to an extracellular form withM _r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

Isozyme polymorphism ofβ-glucosidase inAspergillus nidulans

Electrophoretic analysis of the distribution of various electromorphs at different -glucosidase zones was carried out in natural populations ofA. nidulans, theA. nidulans group, and various species belonging to the genusAspergillus from diverse geographical areas of India. The data show the existence of three segregating zones for -glucosidase, designated -GluI, -GluII and -GluIII. All three zones are present in wild isolates ofA. nidulans, and only two, i.e., -GluI and -GluIII, in theA. nidulans group and -GluII and -GluIII in different species ofAspergillus exceptA. terreus, A. flavus, andA. brevipes, where only -GluIII is present. Overall nine electromorphs are observed at -GluI, three at -GluII, and six at -GluIII zones, respectively, It can be concluded that there may be three structural genes for -glucosidase coding the three polymorphic zones inA. nidulans.This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Synthesis of β(1–2)glucan in Rhizobium loti

Fast growing strains of Rhizobium loti isolated from nodules of Lotus tenuis of the flooding Pampas of Argentina produced cellular (1–2)glucans having a higher degree of polymerization and more anionic substituents than (1–2)glucans accumulated by Agrobacterium tumefaciens cells. Inner membranes of R. loti contained a 235 kDa (1–2)glucan intermediate protein indistinguishable by polyacrylamide gel electrophoresis from the intermediate protein present in A. tumefaciens inner membranes. Incubation of inner membrannes of R. loti with UDP-Gle led to the formation of neutral (1–2)glucans with a higher degree of polymerization than glucans formed by A. tumefaciens inner membranes.Introduction in R. loti strains of plasmid pCD523 containing A. tumefaciens chvA and chvB virulence regions yielded strains that accumulated 4 times more cellular (1–2)glucans than wild type cells. This glucan was, regarding anionic substitution and degree of polymerization, indistinguishable from A. tumefaciens (1–2)glucans. Furthermore inner membranes of these R. loti exoconjugant cells contained higher levels of the 235 kDa (1–2)glucan intermediate protein and formed in vitro 8 times more neutral (1–2)glucan with a degree of polymerization corresponding to A. tumefaciens (1–2)glucan than inner membranes isolated from wild type cells.It was concluded that A. tumefaciens chvB gene is expressed in R. loti and determined the degree of polymerization of (1–2)glucan.Abbreviations Nod⁺ effective nodulation - Vir⁺ virulent - Vir^- avirulent - Trp^r trimethoprim resistence - Tc^r tetracycline resistence - TCA trichloroacetic acid - PMSF phenyl methyl sulfonyl fluoride     

Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta): stabilization,purification, and properties

Adenosine 5-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel-filtration. The native M_r measured by gel-filtration was 350 000. The subunit M_r was estimated to be 43 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In addition, APSSTase had a relatively broad pH optimum of pH 9.0–9.8 with a peak at pH 9.5. The apparentK _m value for adenosine 5-phosphosulfate (APS) was 2.1 M, when dithiothreitol was acceptor substrate. 3-Phosphoadenosine 5-phosphosulfate and inosine 5-phosphosulfate could not substitute for APS as a sulfate donor. The enzyme utilized several organic thiols as acceptor substrates (artificial substrates): dithiothreitol (apparentK _m = 1.5 mM) and dithioerythritol (apparentK _m = 1.5 mM) gave the highest activity, and appreciable activity was also obtained usingl-glutathione (reduced form) which exhibited slight substrate inhibition (apparentK _m = 0.6 mM; the initial velocity was maximal at 3.0–4.0 mM). While APSSTase was markedly unstable in vitro: the half-life for activity loss at 25°C and pH 9.5 was about 8 min, the instability was decreased in the presence of a relatively high concentration of Na₂SO₄ or (NH₄)₂SO₄, and in the presence of APS or its analogs (AMP and -methylene-APS). Most of the thiols, with the sole exception of glutathione, were found to inactivate APSSTase irreversibly. The thiol-mediated inactivation was completely inhibited by the high concentration of Na₂SO₄, and by the analogs of APS.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - -m-APS -methylene-adenosine 5-phosphosulfate - DTT dithiothreitol - IPS inosine 5-phosphosulfate - PAPS 3-phosphoadenosine 5-phosphosulfate We wish to thank Mr. I. Kashiwase, Mr. Y. Endo and Mr. Y. Mimura, School of Fisheries Sciences, Kitasato University, for their technical assistance in this study. The research described in this paper was partly supported by the Kitasato Research Grant (H5-9 and H6-13 to N.K.).     

A highly-sensitive rice seedling bioassay for the detection of femtomole quantities of 3β-hydroxylated gibberellins

A very sensitive and specific bioassay using prohexadione calcium [BX-112, which blocks 2- and 3-hydroxylation of gibberellins (GAs)] with uniconazole (which blocks oxidation of ent-kaurene, ent-kaurenol and ent-kaurenal) in a microdrop assay was developed for several rice (Oryza sativa L.) varieties, including cv. Waito-C, which is already specific to 3-hydroxylated GAs. The sensitivity and specificity of cvs. Waito-C, Tan-ginbozu and Koshihikari to 3-hydroxylated GAs was greatly enhanced by treatment of the seeds with a combination of 40 mM prohexadione calcium and 80 M uniconazole. The minimum detectable doses of 3-hydroxylated GAs (GA₁, GA₃, GA₄ and GA₇) in the three cultivars treated with both chemicals were 1 to 10 fmol (i.e. ca. 350 fg to 3.5 pg) per plant. This is equal to 30-fold more sensitive than Waito-C treated with uniconazole alone, and 30 to 1000-fold more sensitive than Waito-C with no growth retardant soak. Minimum detectable doses of 3-nonhydroxylated GAs (GA₉, GA₁₉ GA₂₀) and GAs with very low biological activity (GA₈ and GA₁₇) were equal to or more than 1000 fmol per plant. This is about equal to the activity in Waito-C treated with uniconazole alone. Application of this assay to an extract from Raphanus sativus was compared with the data by gas chromatography/mass spectrometry (GC/MS), confirming the conclusions reached using authentic test GAs, namely that use of uniconazole plus BX-112 appreciably enhanced the detection sensitivity to fractions shown by GC/MS to contain GA₁ and GA₄, both 3-hydroxylated GAs.Abbreviations GA gibberellin - BX-112 prohexadione calcium     

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