The Structure of Legumin of Vicia faba L.--a Reappraisal

Two-dimensional electrophoretic studies have shown a wide rangeof heterogeneity in the subunits of legumin isolated from seedsof Vicia faba L. As many as 10 disulphide-linked subunit pairsin the mol. wt. range 37 000–79 000 have been observed.Each subunit pair separated on reduction by 2-mercaptoethanolinto a large acidic and a small basic subunit, each of whichwas shown to be heterogeneous in charge by isoelectric focusing.More heterogeneity was found in the large subunits (mol. wt.range 23 000–58 000; pl range 4.6–6.1) than in thesmall subunits (mol. wt. range 21 000–23 000; pl range8.2–8.5). Most legumin molecules seemed to be formed byrandom association of subunit pairs, although one subunit pairassociated only with itself to give a molecular type separableunder non-dissociating conditions.     

Production of Cellulolytic Enzymes by Botryodiplodia theobromae, Pat.

Carboxymethylcellulase (CM-cellulase) and ß-glucosidaseactivities were induced in cultures of Botryodiplodia theobromae,Pat. Both growth of the fungus and CM-cellulase production werebetter with sodium nitrate as nitrogen source than with eitherammonium nitrate or ammonium chloride. Growth, per unit nitrogensupplied, was greater with glutamic acid and aspartic acid asnitrogen sources than with sodium nitrate; this was probablybecause the fungus utilized the carbon of these amino-acids.Most ß-glucosidase activity was formed with ammoniumchloride or ammonium nitrate as nitrogen source. Glucose inhibited the activity of ß-glucosidase markedly.Low glucose concentration (c. 0.003 M) stimulated CM-cellulaseactivity but concentrations above 0.05 M inhibited. The formationof both enzymic activities was repressed in the presence ofglucose. ß-glucosidase activity was more thermolabilethan that of CM-cellulase.     

Studies on the {beta}-glucosidase System of Trifolium repens L.

Using DEAE-cellulose two distinct ß-glucosidase enzymeshave been identified in white clover; one form is primarilyassociated with the seed, the other with young leaves. Evidenceis presented for the separate nature of ß-glucosidaseand ß-galactosidase activity in dry seed flour andin young leaf tissue. These results are considered in relationto the function of the Li locus in white clover, which is shownto control both ß-glucosidase and ß-galactosidaseactivity in expanding leaves.     

Distribution of Glycosidases and Acid Invertase Activities in Relation to Elongation Growth in Pearl Millet Internode

The mean cell length along a differentiating internode and alliedchanges in the activities of ß-glucosidase, - andß-galactosidase. -mannosidase and acid invertase,together with the contents of reducing and non-reducing sugars,were examined in pearl millet (Pennisetum americanum L. Leekecv. BJ-104). The specific activities of cytoplasmic -mannosidase,wall ß-glucosidase, and cytoplasmic and wall acidinvertase showed close relationships with the rate of cell elongation.The linear regressions of the rate of cell elongation, and thespecific activities of wall ß-glucosidase and cytoplasmicand wall invertase showed significant positive correlations(P<0·05), whereas cytoplasmic -mannosidase was negativelycorrelated (P<0·01). The results are discussed in the light of cell wall looseningand the provision of carbon substrates for cell elongation. Key words: Glycosidases, acid invertase, sugars, cell elongation, Pennisetum americanum L., Leeke     

The r{beta}-Conglycinins of Soybean Remain Assembled in a 7S Conformation While Undergoing Proteolytic Cleavage During Germination and Early Seedling Growth

The degradation of the rß-conglycinins, the secondmost abundant seed storage protein complex of Glycine max, thatoccurs as a result of proteolysis during seed germination andearly seedling growth, was investigated. The rß-conglycininsof soybean are composed of a semi-random association of threedifferent subunits, a', a, and rß, in a trimeric complexwith a sedimentation coefficient of 7S. Western immunoblot analysisof the degradation products showed that proteolytic cleavageyields specific fragments as has been shown in other legumes.The proteolytic fragments produced in G. max, cv. Provar aredesignated here as FPI (62 kDa), FPII (57 kDa), FPIII (52 kDa),FPIV (31 kDa), and FPV (27 kDa). Comparison of the fragmentsfrom G. max cv. Keburi, a variant lacking the a' subunit, withthose from G. max cv. Provar showed that the FPIV fragment isderived from the a' subunit. All fragments stained with periodicacid-Schiff's reagent, indicating that exhaustive deglycosylationof these subunits is not a prerequisite for cleavage. All ofthe fragments detected in these experiments sediment in linearsucrose density gradients with sedimentation coefficients ofabout 7S, identical to that of intact rß-conglycinin.These results suggest that as proteolysis of the rß-conglycininsoccur during germination and early growth, the cleavage productsare retained within the holoprotein structure. Further proteolysiscleaves the polypeptides into smaller fragments leading to thedisintegration of the 7S storage protein structure.     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

Physiological and Biochemical Changes Associated with Cotton Fibre Development. IV. Glycosidases and {beta}-1,3-Glucanase Activities

Developing cotton fibre was analysed from 12 days post anthesis(DPA) till maturity for the activity of wall degrading enzymes,ß-galactosidase, ß-glucosidase, -mannosidaseand ß-1,3-glucanase. Each enzyme was estimated inthree different fractions namely cytoplasmic, ionically wall-boundand covalently wall-bound. There was a significant correlationbetween ß-galactosidase and ß-glucosidaseactivities in the covalently bound fraction, and the rate offibre elongation. Similarly, covalently bound ß-1,3-glucanaseactivity showed an increasing trend up to 18 DPA, i.e. aboutthe time when maximum rate of fibre elongation was achieved. The results presented here suggest that covalently wall-boundglycosidases may have an importafit role in cell wall loosening.Earlier reports providing evidence against the involvement ofthese enzymes in elongation growth in intact system, may perhapsbe due to scant attention paid to the subcellular distributionof these enzymes. Gossypium hirsutum, cotton fibre, glucanase, glycosidase, wall-loosening     

Development of {beta}-amylase activity and polymorphism in wheat seedling shoot tissues8

Increasing ß-amylase activity in wheat (Triticum aestlvum,var. Star) seedling shoot tissues was consistently accompaniedby the development of a characteristic polymorphism of the enzyme,as shown by electrophoresis employing amylopectin-containingpolyacrylamide gels. Very young shoot tissue contained one principalform of the enzyme (ß1), whereas two other major forms(ß2, ß3) appeared complementary to thisupon further growth. In vitro incubation experiments indicatedthat the polymorphism arose via a probably proteolytic conversionof ß1 into ß2 and ß3. The conversioninvolved neither an activation of ß-amylase nor asignificant modification of ß-amylase component plvalues. The electrophoretic ß-amylase patterns ofsubcellular leaf compartments suggested that ß1 issynthesized in the cytoplasm of leaf mesophyfi cells and thatthe other forms arise upon transfer of this ‘primary’form into the vacuole. The development of shoot ß-amylaseactivity did not require light, but appeared to be under thenegative control of the chloroplast and was stimulated by mineralnutrients. No clear relationship between ß-amylaseactivity and starch metabolism was evident, since the leaf activitywas largely absent from mesophyll protoplasts, could not beunequivocally demonstrated in the mesophyll chioroplasts, anddeveloped regardless of whether the tissues contained significantamounts of starch or not. Key words: Wheat, leaves, ß-amylase, polymorphism, compartmentation     

Injury-induced Cyanogenesis in Vegetative and Reproductive Parts of Two Grevillea Species and their F1 Hybrid

When leaves of three Grevillea species were macerated, hydrogencyanide was only released from the soft, immature ones. Thebark but not wood of young stems was weakly cyanogenic. Thegreatest concentration of cyanoglucosides occurred in peduncles,flower buds, open flowers, immature seeds and germinants. Nocyanide was released from nectar, pedicels, dry fruit, elaiosomesand dry walls mature seeds. Addition of ß-glucosidase,and sometimes water only, increased cyanogenesis in young leaves,bark, some seed and fruit walls, and embryos of mature seeds.The endogenous levels of ß-glucosidase were sufficientto release all the cyanide present in at least 80% of cases. Young leaves and germinants of G. bipinnatifida (resprouter)released more cyanide than those of G. banksii, while flowerbuds, stigmatic region, styles, perianths and ovaries of G.banksii (non-sprouter) released far more cyanide than thoseof G. bipinnatifida. The interspecific and intraplant distributionof cyanoglucosides is consistent with the expected impact ofbeneficial and herbivorous animals on long term fitness of thesespecies. The F1 hybrid (G. 'Robyn Gordon') tended to have intermediatecyanoglucoside levels between the two putative parents.Copyright1993, 1999 Academic Press Grevillea bipinnatifida, G. banksii, G. 'Robyn Gordon', cyanide, herbivory, ß-glucosidase, cyanogenesis     

A novel {beta}-galactosidase gene isolated from the bacterium Xanthomonas manihotis exhibits strong homology to several eukaryotic {beta}-galactosidases

The gene encoding a ß-galactosidase from Xanthomonasmanihotis was cloned into Escherichia coli. The gene resideson a 2.4 kb DNA fragment which was isolated from a partial Sau3Alibrary in the cloning vector pUC19 using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as the selection. The enzyme produced by the clone hasa specificity for ß1-3->ß1-4-linked galactose.The nucleotide sequence of the gene was determined. The deducedprotein sequence contained 597 amino acids yielding a monomericmolecular mass of 66 kDa. The cloned ß-galactosidaseshowed no similarity to any known prokaryotic ß-galactosidase.However, extensive similarity was observed with eukaryotic ß-galactosidasesfrom animals, plants and fungi. The strongest similarity waswith the ß-galactosidases found hi the human and mouselysosomes (42 and 41% identity, respectively). Alignment ofthe X.manihotis and eukaryotic ß-galactosidase sequencesrevealed seven highly conserved domains common to each protein.Additionally, Domain 1 in X.manihotis showed similarity to regionswithin catalytic domains from seven xylanases and cellulasesbelonging to family 10 of glucosyl hydrolases. A region spanningDomain 2 showed similarity to the catalytic domain of endo ß1-3glucanases from tobacco and barley. cellulase ß-galactosidase G_M$$$gangliosidosis Morquio B syndrome Xanthomonas     

Glycoprotein Nature of Glycosidases from Leaves of Pisum sativum L

-Mannosidase, ß-N-acetylglucosaminidase, - and ß-galactosidaseand ß-glucosidase were partially purified from leavesof Pisum sativum by ammonium sulphate fractionation and columnchromatography on DEAE-Sephadex A-50 and hydroxylapatite. Atleast two molecular forms of each enzyme were resolved by thesetechniques except for ß-glucosidase of which onlyone form was resolved. Except for one form of -galactosidase,all of the glycosidases thus purified were completely boundby Sepharose-linked Concanavalin A. The binding was stronglyinhibited by cr-methyl-D-mannoside and no binding to Sepharose-6-Boccurred indicating that these glycosidases contain mannose-richoligosaccharides. The glycoprotein nature of -mannosidase, ß-galactosidaseand ß-glucosidase was further demonstrated by chromatographyon phenylboronate agarose columns. The differences in the concentrationof cr-methyl-D-mannoside and sorbitol required to elute thevarious glycosidases from Sepharose-linked Con A and phenylboronateagarose, respectively, suggested that these enzymes are glycosylatedto various degrees or that structural variation in their carbohydratemoieties occur. This is the first demonstration that glycosylationof several glycosidases present in a single plant species isapparently a generalized feature of these enzymes. Key words: Pisum sativum, Glycosidase, Glycoprotein     

Extraction of Enzymes from Cell Walls of Sugar Beet Cells Grown in Suspension Culture

Various glycosidases were extracted from cell walls of sugarbeet cells grown in suspension culture using three successiveprocedures employing saline, EDTA, and commercial cellulase.Saline was effective for extracting acid invertase, ß-galactosidase,and ß-glucosidase. EDTA extracted most of the -galactosidaseand some of the ß-glucosidase. It was most effectivebetween 27?C and 40?C. Commercial cellulase could extract mostof the -mannosidase in the cell wall when used at 27?C for 96h. These three procedures could not extract some of the acidinvertase and ß-glucosidase. The results suggest thatthe cell wall glycosidases are associated with different polysaccharides. These extraction procedures were also applied to the cell wallsof intact tissues, such as cotyledons, hypocotyls plus roots,and mature roots of sugar beets. EDTA as well as saline wasquite effective for extracting bound enzymes from the cell wallof intact tissue, which indicated that extraction with EDTAis useful for liberating bound enzymes from plant cell walls. (Received October 19, 1987; Accepted March 16, 1988)     

Molecular and Subunit Heterogeneity of Legumin of Pisum sativum L. (Garden Pea)-- A Multi-Dimensional Gel Electrophoretic Study

Pisum sativum legumin has been shown to be heterogeneous withrespect to the existence of different molecular forms, the molecularweights of subunit pairs, and the molecular weights and isoelectricpoints of constituent subunits. Disulphide-bonded subunit pairswith mol. wt. 54 000 constitute a major part of total legumin.In addition, subunit pairs varying in mol. wt. from 35 000 to58 000 have been observed which associate in various ways togive rise to at least three distinct molecular forms of leguminseparable on non-dissociating gels. ‘Three-dimensional’gel electro-phoresis has been employed to understand the consitutionof these forms in detail.     

Comprehensive Enzymatic Analysis of the Cellulolytic System in Digestive Fluid of the Sea Hare Aplysia kurodai. Efficient Glucose Release from Sea Lettuce by Synergistic Action of 45 kDa Endoglucanase and 210 kDa ?-Glucosidase

Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.     

Molecular Cloning and Characterization of a cDNA for the r{beta} Subunit of a G Protein from Rice

We isolated a cDNA for the rß subunit of a heterotrimericG protein from rice (Oryza sativa L. cv. Nipponbare). The aminoacid sequence deduced from the cDNA was 76% and 94% homologusto the sequences of the rß subunits from Arabidopsisand maize (AGrß1 and ZGrß1), respectively. (Received July 28, 1995; Accepted December 25, 1995)     

r{beta}-Glucosidase in the Indigo Plant: Intracellular Localization and Tissue Specific Expression in Leaves

rß-Glucosidase of indigo plant (Polygonum tinctorium)has a high substrate specificity for indican (indoxyl rß-D-gIu-coside).To examine the localization of this rß-glucosidase,we fractionated the cells of the leaves and analysed them im-munocytochemically.Immunoelectron micrographs with specific antibodies againstthe rßglucosidase clearly showed that the rß-glucosidasewas localized in the stroma of the chloroplasts in mesophyllcells, but not in the thylakoid membrane. Chloroplasts wereisolated from the crude ho-mogenate of the fresh leaves by Percolldensity gradient centrifugation and then subjected to suborganellarfrac-tionation. rßGlucosidase activity was specificallydetected in the stromal fraction, but not in the thylakoid membrane.This was also supported by the result of an immunoblot of thefractions with anti-rßglucosidase antibodies. Therß-gIu-cosidase was immunocytochemically localizedin the chloroplasts of mesophyll cells, but not in any chloroplastsin marginal cells of the vascular bundle or epidermal cells;ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromalprotein, was observed in all chloroplasts in these cells. Theseresults suggest that rß-glucosidase is tissue specificin its expression in the leaves of the indigo plant. (Received April 14, 1997; Accepted July 10, 1997)     

Characterization of Common Carbohydrate Antigenic Determinants on Soya Bean Cell-Wall Enzymes

Three soya-bean (Glycine max) cell-wall enzymes (ß-glucosidase,pectin methyl esterase and phosphatase) have been found to beglycoproteins. The polyclonal antibodies raised against pectinmethyl esterase and ß-glucosidase lacked specificity,cross-reacted highly with native enzymes and also both reactedwith pure soya-bean phosphatase, horseradish peroxidase andhoneybee venom phospholipase A2. They did not react with eithernon-glycosylated bacterial phosphatase or deglycosylated cell-wallenzymes. The two antisera contained both non-specific anti-glycanantibodies and specific anti-polypeptide antibodies that werequantified. Antiglycan antibodies specific to 1–3 fucoseand ß1–2 xylose were detected in both antiseraand were separated and quantified. The occurrence of terminalfucose (and mannose) was confirmed with specific lectins. Theseresults indicate that most of the common glycan epitopes probablycorrespond to the asparagine-linked complex glycan previouslydetected in several glycoproteins of plants as well as in thoseof molluscs and insects. (Received March 10, 1993; Accepted November 5, 1993)     

Characterization in cultured cerebellar granule cells and in the developing rat brain of mRNA variants for the NMDA receptor 2C subunit

N-Methyl-D-aspartate (NMDA) receptors are heteromeric structures resulting from the association of at least two distantly related subunit types, NR1 and one of the four NR2 subunits (NR2A-NR2D). When associated with NR1, the NR2 subunits impose specific properties to the reconstituted NMDA receptors. Although the NR1 mRNAs are expressed in the majority of central neurons, the NR2 subunits display distinct patterns of expression in the developing and adult rat brain. The NR2C subunit is barely expressed in the rat forebrain, whereas its expression increases substantially in the granule cells in the course of cerebellar development. We have identified novel NR2C splice variants in cultured cerebellar granule cells as well as in the developing cerebellum. When compared with the prototypic NR2C mRNA, these variants carry one (NR2Cb) or two (NR2Cd) insertions or a deletion (NR2Cc) and encode putative NR2C polypeptides that terminate between the third and fourth membrane segments or between the first and second membrane segments. RT-PCR analysis and in situ hybridization show that expression of the splice variants is developmentally regulated, both in the cerebellum and in the hippocampus. Electrophysiological recordings and microfluorimetry emissions in transfected human embryonic kidney 293 cells indicate that the NR2Cb variant, when expressed in combination with NR1, does not contribute to the formation of functional receptor channels. The significance of theses findings is discussed.