The Oligosaccharide Units of the Xyloglucans in the Cell Walls of Bulbs of Onion, Garlic and their Hybrid

Xyloglucans were isolated from the 24% KOH-soluble fractionof the cell walls of bulbs of onion (Allium cepa), garlic (Alliumsativum) and their hybrid. The polysaccharides yielded singlepeaks upon gel filtration with average moleular weights of 65,000for onion, 55,000 for garlic and 82,000 for the hybrid. Compositionalanalysis of the oligosaccharide units after digestion with anendo-1,4-ß-glucanase from Streptomyces indicated thatthe polysaccharides were constructed of four kinds of repeatingoligosaccharide unit, namely, a decasaccharide (glucose/xylose/galactose/fucose,4 : 3 : 2 : 1), a nonasaccharide (glucose/xylose/galactose/fucose,4 : 3 : 1 : 1), an octasaccharide (glucose/xylose/galactose,4 : 3 : 1 ) , and a heptasaccharide (glucose/xylose, 4 : 3).The xyloglucan from the hybrid contained highly fucosylatedunits that resembled those from onion rather than from garlic.The analysis also revealed that the xyloglucans from Alliumspecies contain highly substituted xylosyl residues with fucosyl-galactosylresidues, suggesting that these monocotyledonous plants resembledicotyledons in the structural features of their xyloglucans. (Received November 1, 1993; Accepted June 16, 1994)     

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     

A Further Study of {beta}-Galactosidase Activity in Apples Ripening in Store

Apples (Malus domestica Borkh.) were ripened in 2% O₂ at 3·3°C. The loss of galactose residues from the cortical cellwalls was initiated after 27 d although an increase in the ß-galactosidaseactivity of the tissue did not occur until after 50 d. ß-Galactosidase activity associated with the cellwall was determined using discs of cortical tissue. A substantiallevel of activity was observed prior to picking and subsequentlyduring ripening of the apples in air.     

Inhibition of Zeatin-Induced Growth of Cucumber Cotyledons by Sulfite Ions

The inhibitory effects of sulfite ions on zeatin-induced cellexpansion in cotyledons excised from dark-grown seedlings ofcucumber (Cucumis sativus L.) were examined. With 50 µMzeatin the growth rate in white light was about twice that ofthe control. Addition of Na₂SO₃ in the growth medium inhibitedthe zeatin-induced growth of cotyledons. Zeatin-treatment increasedthe osmotic potential in cell sap of cotyledons, while sulfitedecreased it. These treatments had no significant effect onpotassium concentration. Sulfite inhibited the zeatin-inducedincrease in contents of fructose and glucose, but did not affectsucrose content. The relative contents of non-cellulosic constituentsof cell walls fell with the advance of culture. This decreasewas repressed by sulfite, indicating that inhibition of expansiongrowth in cucumber cotyledons by sulfite ions was the resultof alterations in the cell wall structure due to changes inthe cell wall metabolism. (Received June 12, 1984; Accepted October 24, 1984)     

Evidence for Several Galactan Synthases in Flax (Linum usitassimum L.) Suspension-Cultured Cells

A membrane fraction from flax cells was able to incorporate[¹⁴C]galactose from UDP-D-[¹⁴C]galactose in vitro. The productsof the reaction, characterized by methylation analysis, consistedof a rß-1,4-galactan (solubilized mainly in water)and a rß-1,3- rß-1,6-galactan (solubilizedmainly in alkali). These results indicated the presence of severalgalactan synthase complexes, as did a profile of the relationshipbetween pH and activity which revealed both a maximum at pH6.5 and a shoulder at pH 8. Moreover, galactan synthase activitieswere found at two densities: 1.125 g cm^–3 (Golgi membranes)and 1.07–1.08 g cm^–3 (corresponding to low-densityvesicles). Partial characterization of one enzymatic system (maximaly activeat pH 8 in the presence of 5 mM MgCl₂) was achieved. The K_mfor UDP-galactose and V_max were 38 µM and 4.5 nmol h^–1(mg protein)^–1, respectively. (Received June 6, 1993; Accepted September 22, 1993)     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

Auxin-Induced Changes in the Cell Wall Xyloglucans: Effects of Auxin on the Two Different Subtractions of Xyloglucans in the Epicotyl Cell Wall of Vigna angularis

In order to study the IAA-induced modifications of the cellwall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara)epicotyl segments, the xyloglucans were subfractionated intotwo components, i.e., 4K-U and 24K xyloglucans, which were obtainedby extraction with 4% KOH solution containing 8 M urea and 24%KOH solution, respectively. The weight-average molecular weightsof 4K-U and 24K xyloglucans were estimated to be 40 x 10⁴ and106 x 10⁴, respectively. Complete acid hydrolysis of 4K-U and24K xyloglucans gave glucose, xylose, galactose and fucose inmole % 48.3 : 33.5 : 13.8 : 4.4 and 45.3 : 30.9 : 19.6 : 4.3,respectively. Treatment of epicotyl segments with IAA (0.1 mM) caused a decreasein the amount of 24K xyloglucans and an increase in 4K-U xyloglucans,whereas the total amount of the two xyloglucans remained constant.Furthermore, IAA treatment caused a decrease in the molecularweight of 24K xyloglucans from 106 x 10⁴ to 78 x 10⁴ withoutcausing changes in their sugar compositions. With 4K-U xyloglucans,IAA caused an increase in the mole % of xylose and a decreasein the mole % of galactose and fucose. ¹ This paper is dedicated to the late Professor Joji Ashida. (Received November 26, 1982; Accepted February 7, 1983)     

Human serum contains a chitinase: identification of an enzyme, formerly described as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase)

Since 1988 an endoglucosaminidase, provisionally named MU-TACThydrolase, has been known that hydrolyses the artificial substrate4-methylumbelliferyl-tetra-N-acetyl-chitotetraoside (MU-[GlcNAc]₄,where GlcNAc is N-acetyl-glucosamine). The biological functionof the enzyme was unknown. In this paper evidence is presentedshowing that this endoglucosaminidase from human serum is infact a chitinase that is different from lysozyme. The factssustaining this finding are: (i) the identification of the productsformed from MU-[GlcNAc]₃ as [GlcNAc]₂ and [GlcNAc]₃; (ii) chitinand ethylene glycolchitin can be degraded by the enzyme; (iii)the chitinase inhibitor allosamidin also inhibits the actionof MU-TACT hydrolase from human serum; (iv) no hydrolysis ofthe lysozyme substrate Micrococcus lysodeikticus. The enzymealso occurs in rat liver. It was demonstrated that upon Percolldensity gradient centrifugation the enzyme from this tissuedistributed parallel to the lysosomal marker enzymes ß-N-acetylhexosaminidaseand ß-galactosidase, indicating a lysosomal localizationfor this enzyme. It is proposed that the enzyme functions inthe hydrolysis of chitin, to which mammals are frequently exposedduring infection by pathogens. allosamidin chitinase human serum lysozyme MUTACT hydrolase     

Comparative Studies of Acid Glycosidases from Three Legumes

The major isoenzymes of -mannosidase (EC 3.2.1.24 [EC] ) and ß-galactosidase(ECf 3.2.1.23 [EC] ) have been separated from cotyledons of gardenpea, Pisum sativum L. (Vicieae), chick pea, Cicer arietinumL. (Cicereae), and cowpea, Vigna unguiculata (L.) Walp. (Phaseoleae).Some of their properties have been determined, including pHoptima, K_m values for p-nitrophenyl glycosidc substrates, andthe effects of several inhibitors. Swainsonine, an indolizidinealkaloid, was the most effective inhibitor of mannosidase 1,with I₃₀ values of 5.6 x 10^–8 M (cowpea), 1x 10^–7M (chick pea) and 2.9 x 19^–7 M (pea). The most effectiveinhibitor of ß-galactosidase 2 from all sources wasD-galactonic acid-1,4-lactonwe (-lactone), with K_i values rangingbetween 3.0 and 3.9x 10^–3 M. An inhibitor of the E. coliß-galactosidose, p-aminophenyl thio-ß-D-galactopyranoside,did not inhibit any of the legume ß-galctosidases;rather it enhanced the activites of the enzymes from chick peaand cowpea cotyledons. Etiolated hull and seed tissues frompea pods developing in darkness contained similar acid glycosidaseactivities to normal green tissues, thus the chloroplast isan unlikely location for ß-galactosidase 2. The majorß-galactosidasesdetected with an indigogenic substrate (5-bromo-4-chloro-3-indoxyl-ß-D-galactopyranoside)following gel electrophoresis of extracts from pea hull, seedcoats and cotyledons appeared to be different from ß-galactosidase2. Acid glycosidase, cotyledon, isoenzyme, -lactone, legume, swainsonine     

Levels of {beta}-Glucan and Lignin in Elongating Internodes of Deepwater Rice

Partial submergence or treatment with either ethylene or gibberellicacid (GA₃ induces rapid growth in deepwater rice (Oryza sativaL.). We correlated the synthesis of two cell wall componentswith two phases of internodal elongation, namely (13,14)-ß-glucanformation with cell elongation and lignification with differentiationof the secondary cell wall and cessation of growth. The contentof ß-glucan was highest in the zone of cell elongationin internodes of air-grown plants and plants that were inducedto grow rapidly by submergence. In the intercalary meristemand in the differentiation zone of the internode, ß-glucanlevels were ca. 70% lower than in the zone of cell elongation.The outer cell layers, enriched in epidermis, contained moreß-glucan in submerged, rapidly growing internodesthan in air-grown, control internodes. The ß-glucancontent of the inner, parenchymal tissue was unaffected or slightlylowered by submergence. The epidermis appears to be the growth-limitingstructure of rapidly growing rice internodes. We hypothesizethat elevated levels of ß-glucan contribute to elongationgrowth by increasing the extensibility of the cell wall. Lignificationwas monitored by measuring the content of lignin and the activitiesof two enzymes of the lignin biosynthetic pathway, coniferylalcohol dehydrogenase (CAD) and phenylalanine ammonia-lyase(PAL), in growing and non-growing regions of the internode.Using submerged whole plants and GA₃-treated excised stem segments,we showed that lignin content and CAD activity were up to sixfoldlower in newly formed internodal tissue of rapidly growing ricethan in slowly growing tissue. No differences were observedin parts of the internode that had been formed prior to inductionof growth. PAL activity was reduced throughout the internodeof submerged plants. We conclude that lignification is one ofthe processes that is suppressed to permit rapid growth. ¹ This work was supported by the National Science Foundationthrough grants No. DCB-8718873 and DCB-9103747 and by the Departmentof Energy through grant No. DE-FGO2-90ER20021. M.S. was therecipient of a fellowship from the Max Kade Foundation.     

Effects of a Plant-Growth Regulator, Prohexadione, on the Biosynthesis of Gibberellins in Cell-Free Systems Derived from Immature Seeds

Inhibition of the biosynthesis of gibberellins by prohexadione,3,5-dioxo-4-propionylcyclo-hexanecarboxylic acid, was studiedwith cell-free systems derived from immature seeds of Cucur-bitamaxima, Phaseolus vulgaris and Pisum sativum. Prohexadione,at a concentration of 10–⁴ M, inhibited C-7 oxidationof GA₁₂-aldehyde, C-20 oxidation of GA₁₅, conversion of C₂₀-gib-berellinsto C₁₉-gibberellins, 3ß-hydroxylation, 2,3-dehydrogenationof GA²⁰, 2,3-epoxidation of GA₅ and 2ß-hydroxylationof GA₉ and GA₂₀. The 3ß-hydroxylase activity appearedto be more sensitive to prohexadione than were the C-20 oxygenaseand the 2ß-hydroxylase activities. The conversionof mevalonic acid to GA₁₂-aldehyde and the 13-hydroxylationof GA₁₂ were not affected by prohexadione at a concentrationof 3 ? 10^–4 M. All of the steps inhibited by prohexadioneare oxidation steps catalyzed by soluble enzymes that require2-oxoglutarate, Fe²⁺ and oxygen, and all of them occur distalto the synthesis of GA₁₂-aldehyde in the biosynthesis of gibberellins. (Received April 4, 1990; Accepted September 14, 1990)     

Crystalline Cellulose in Hydrated Primary Cell Walls of Three Monocotyledons and One Dicotyledon

The molecular ordering of cellulose, including its crystallinity,in the unlignified primary cell walls of three monocotyledons(Italian ryegrass, pineapple, and onion) and one dicotyledon(cabbage) was characterized by solid-state ¹³C NMR spectroscopy.These species were chosen because their primary cell walls havedifferent non-cellulosic polysaccharides and this may affectthe molecular ordering of cellulose. Values of the proton rotating-framerelaxation [T_1p(H)] and spin-spin relaxation [T₂(H)] time constantsshowed that the cellulose in the cell walls of all four specieswas in a crystalline rather than an amorphous state. Furthermore,a resolution enhancement procedure showed that the triclinic(I) and the monoclinic (I_rß) crystal forms of cellulosewere present in similar proportions in these cell walls. However,the calculated cross-sectional dimensions of the cellulose crystallitesvaried among the cell walls (in the range 2–3 nm): thelargest were in the Italian ryegrass, the smallest were in theonion and cabbage, and those of intermediate size were in thepineapple. The crystallite dimensions may thus be affected bythe non-cellulosic polysaccha-ride compositions of the cellwalls. ⁴Present address: Food Science Postgraduate Programme, Departmentof Chemistry, The University of Auckland, Private Bag 92019,Auckland, New Zealand.     

Changes in Cell Wall Composition and Water-soluble Polysaccharides During Kiwifruit Development

Changes in both cell wall and water-soluble polysaccharide compositionduring the growth of kiwifruits [Actinidia deliciosa (A. chev.) C. F. Liang and A. R. Ferguson var. deliciosa ‘Hayward’]were investigated. Cellulose was the major wall polysaccharide,with galactose and uronics the main non-cellulosic sugars. Muchsolubilization of cell wall pectic polysaccharides was detected.While wall-galactose solubilization started 3 months after anthesis,polyuronide degradation did not start until the fifth month,1 month prior to the harvest date. Parallel to these processes,a linear increase in water-soluble polysaccharides was detected.These mainly comprised galactose-rich polymers in the first3 months and little-branched polyuronides after the fifth month.Two different mechanisms for galactose and uronic acid solubilizationfrom kiwifruit cell walls during fruit development are proposed. Actinidia deliciosa ; cell wall; fruit growth; kiwifruit; water-soluble polysaccharides     

{beta}-1,4-Glucan occurring in homogenate of Phaseolus aureus seedlings. Possible nascent stage of cellulose biosynthesis in vivo

A small amount of cytoplasmic ß-1,4-glucan, whichmight be involved in the synthesis of cellulose in the cellwall, was found in the homogenate prepared from the hypocotylsof seedlings of Phaseolus aureus. Upon hydrolysis by cellulaseof the 20,000?g pellet from the cytoplasmic fraction of segmentsincubated in a [¹⁴C]-glucose solution, [¹⁴C]-cellobiose wasproduced, with specific radioactivities 3 to 10 times greaterthan those of the cellobiose from cellulose in the cell wallat various incubation periods. The incoporation of radioactivityfrom [¹⁴C]-glucose into this cytoplasmic ß-1,4-glucanwas therefore faster than that into cellulose constituting thecell wall. Hence, it seemed that the former ß-1,4-glucancould be turned over. To examine whether the- cytoplasmic ß-1,4-glucanis carried by some subcellular components, cytoplasmic ß-1,4-glucanin the cell was fractionated by differential centrifugation,two enzyme activities being measured as the markers of subcellularcomponents. The distribution of ß-1,4-glucan was similarto that of UDPG-glucosyltransferase activity but not to thatof IDP-ase activity. The result suggests that the cytoplasmicß-1,4-glucan has some relation to plasma membranes. Coumarin, known as a specific inhibitor for the biosynthesisof cellulose in plant cells, was shown to inhibit the incorporationof radiocarbon from [¹⁴C]-glucose into cytoplasmic ß-1,4-glucanto the same extent as that into cellulose in the cell wall ofthe hypocotyls. ¹ Present address: Department of Biological Science, TohokuUniversity, Kawauchi, Sendai 980, Japan. (Received May 31, 1976; )     

Effects of Deoxygibberellin C (DGC) and 16-Deoxo-DGC on Gibberellin 3{beta}-Hydroxylase and Plant Growth

Deoxygibberellin C (DGC), a C/D ring-rearranged isomer of GA₂₀,was shown to inhibit the conversion of [2,3-³H₂]GA₉ to [2-³H]GA₄by gibberellin 3ß-hydroxylase from immature seedsof Phaseolus vulgahs. Deoxygibberellin C inhibited the promotionof growth by exogenously applied GA₂₀ of rice (Oryza sativaL.) seedlings. Evidence is also presented that DGC is a competitiveinhibitor of the 3ß-hydroxylase from P. vulgaris.However, DGC only weakly inhibited the conversion catalyzedby the 3ß-hydroxylase from Cucurbita maxima at highconcentrations, and it did not inhibit the promotion of growthby exogenously applied GA₉ of cucumber (Cucumis sativus) seedlings.These results suggest that the 3ß-hydroxylases fromP. vulgaris and C. maxima have different structural requirementswith respect to their substrates. 16-Deoxo-DGC also inhibitedcatalysis of the same conversion by 3ß-hydroxylasefrom P. vulgaris, and it slightly inhibited the conversion catalyzedby the enzyme from C. maxima. Application of 16-deoxo-DGC causedthe promotion of the growth of seedlings of both rice and cucumber. ³ Present address: Genetic Engineering Center, Korea Instituteof Science and Technology, Daejeon 305–606, Korea ⁴ Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Utsunomiya-shi, Tochigi, 321 Japan (Received September 25, 1990; Accepted December 17, 1990)     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Effects of Fluorogibberellins on Plant Growth and Gibberellin 3r{beta}-Hydroxylases

3rß-Fluorogibberellin A₉ (3rß-fluoro-GA₉),3rßfluoro-GA₂₀, 3rß-fluorodeoxygibberellinC (3rß-fluoro-DGC) and 13-fluoro-GA₉ were prepared,and their effects on plant growth and gibberellin (GA) 3rß-hydroxyIaseswere examined. 3rß-Fluoro-GA₉ and 3rß-fluoro-GA₂₀promoted the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)seedlings to three times higher than the control seedlings ata dosage of 3 µ plant^–1, and 3rßfluoro-DGCto twice higher at the same dosage. 3rßg-Fluoro-GA₉was active in cucumber (Cucumis sativus L.) hypocotyl assay,its activity being about one-thirtieth as much as that of GA₄.3rß-Fluoro-GAs were active per se in promoting theshoot elongation of rice. 3rß-Fluoro-DGC inhibitedthe 3rß-hydroxylation of [³H₂]GA₉ to [³H]GA₄ by GArß-hydroxylase from bean (Phaseolus vulgaris L.),but 3rß-fluoro-GA₉ and 3rß-fluoro-GA₂₀ didnot show any effects on the enzyme activity. These 3rß-fluoro-GAsalso showed no or only a weak inhibitory effect on the rß-hydroxylasefrom pumpkin (Cucurbita maxima L.). 13-Fluoro-GA₉ promoted growthof rice and cucumber seedlings, and inhibited the 3rß-hydroxylasesfrom both bean and cucumber. 13-Fluoro-GA₉was converted into13-fluoro-GA₄ and 2,3-didehydro-13-fluoro-GA₉, in a cell-freesystem from bean, and conversion of 13-fluoro-GA₉ into 13-fluoro-GA₄was also observed in a cell-free system from pumpkin. Theseresults suggest that 13-fluoro-GA₉ is one of the substratesof GA 3rß-hydroxy-lases, and that 13-fluoro-GA₉ isactive as a result of the conversion to 13-fluoro-GA₄ in riceand cucumber seedlings. (Received October 27, 1997; Accepted March 13, 1998)     

Identification, Purification and Properties of the Precursor of Conglutin {beta}, The 7S Storage Globulin of Lupinus albus L. Seeds

Lupinus albus L. developing cotyledons 35 d after floweringcontained a major polypeptide of-average M_r 64000, immunologicallyrelated to conglutin ß, the 7S storage globulin ofthis seed. This polypeptide decreased during seed maturation,without completely disappearing in the mature seed. This dropwas accompanied by the formation of polypeptide fragments typicalof the mature conglutin ß. The 64000 polypeptide hasbeen identified as the precursor polypeptide of conglutin ß. Undenatured conglutin ß precursor, purified by ionexchange chromatography and size exclusion chromatography, showedsurface and association properties identical to the mature conglutinß molecule. The precursor oligomer, of M_r 190000,consisted of an association of three 64000 subunits. They stronglyreacted with concanavalin A indicating the presence of covalentlylinked carbohydrate. Tryptic treatment of the undenatured conglutin ß precursorled to the accumulation of a relatively stable 59000 polypeptidewhich was cleaved later on and produced three large polypeptidefragments differing from the mature conglutin ß polypeptides. Key words: Conglutin ß, precursor, developing seeds, Lupinus albus L.     

Changes in the composition of cotton fibre cell walls during development

Purified cell walls, prepared from cotton fibres (Gossypium arboreum L.) at different growth stages, were subjected to successive extractions to give pectic, hemicellulosic, and -cellulosic fractions. The protein content and sugars obtained after hydrolysis of the total cell walls and of the various fractions were quantitatively estimated. The amount of protein in the fibre cell walls from one ovule reached a maximum value at the end of the elongation growth, decreased, and then reached a second maximum at the end of the secondary wall deposition. The absolute amounts of fucose, galactose, mannose, rhamnose, arabinose, uronic acid, and non-cellulosic glucose residues all reached a maximum at the end of the primary wall formation or at the beginning of the secondary wall formation. Only the absolute amounts of xylose and of the cellulosic glucose residues increased until the end of the fibre development. Most conspicuous was the decrease in the absolute amounts of non-cellulosic glucose and of arabinose residues during the secondary wall formation, possibly indicating a turnover of at least some of the hemicellulosic wall material.Abbreviations DPA days post anthesis - TLC thin layer chromatography - SDS sodium dodecyl sulphate     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com