The Structure and Functions of Xyloglucan

Xyloglucan is a polysaccharide found in the primary cell wallsof all higher plants examined. Its cellulose-like backbone,which is about 0.15 to 1.5 µm long, consists of 300 to3 000 ß-(14)-linked D-glucopyranose residues. About60–75% (or, in grasses, about 30–40%) of the glucoseresidues have side-chains attached to position 6. The majorside-chains are: D-xylopyranosyl--1 -, D-galactopyranosyl-ß-(12)-D-xylopyranosyl--I , L-arabinofuranosyl-(1 -2)-D-xylopyranosyl--1-, and (except in grasses) L.-fucopyranosyl--(1 -2)-D-galactopyranosyl-ß-(1-2)-D-xylopyranosyl--1-. There is some regularity in the distribution of these side-chainsalong the backbone. Xyloglucan plays two very different r?les in the control ofcell growth: (a) as a major building material of the wall [concentrationof xyloglucan in the wall in vivo 10% (w/v)] it probably directlydictates wall extensibility and, therefore, the rate of cellexpansion and (b) it can be broken down to a fucose-containingoligosaccharide which [at a concentration of 0.0000001% (w/v)]exerts a hormone-like anti-auxin effect on growth. In addition,xyloglucan lacking fucose is used by certain dicotyledonousseeds as a food reserve which is mobilized after germination.Xyloglucan is, therefore, the subject of considerable currentinterest in several apparently disparate areas of botany. Key words: Xyloglucan, ‘oligosaccharin’, hemicellulose, auxin, anti-auxin, growth, cell walls, reserve carbohydrate     

Effects of auxin on the structure of hemicelluloses of Avene coleoptiles

Avena coleoptile hemicelluloses were fractionated into water-solublehemicelluloses I and IIB and water-insoluble hemicellulose IIA.These hemicelluloses were then subjected to glycosidic linkageanalysis by methylation technique, which revealed that hemicelluloseI was predominantly composed of arabinoxylans and ß-(l4)glucans and hemicellulose IIB was composed of arabinoxylans,ß-(l4) : (l3)-mixed linked glucans, ß-(l4)-glucansand xyloglucans. Hemicellulose IIA was mainly composed of xyloglucansand probably ß-(l4)-glucans. Methylation analysisof hemicelluloses extracted from Avena coleoptile segments treatedwith auxin in the presence of mannitol (0.15 M) indicated thatauxin apparently had no effect on the structure of arabinoxylanand caused a specific decrease in the amount of ß-(l4): (l3)-mixed linked glucan. (Received November 19, 1979; )     

Xyloglucan and r{beta}-D-Glucan in Cell Walls of Rice Seedlings

Cell walls of 4-day old rice seedlings were extracted successivelywith ammonium oxalate-oxalic acid, 4% KOH and 24% KOH. A rß-D-glucanpreparation and a xyloglucan preparation were isolated fromthe 4% KOH extract and 24% KOH extract, respectively. Methylationanalysis and enzymic degradation studies of the polysaccharidesshowed that the former was built up predominantly of repeating-oligosaccharideunits of 3-O-rß-cellobiosyl-D-glucose and 3-O-rß-cellotriosyl-D-glucosein a molar ratio of 2.6 : 1.0, and the latter was of repeating-oligosaccharideunits of -D-xylosyl-(16)-rß-D-glucosyl-(14)-[-D-xylosyl-(16)]-rß-D-glucosyl-(14)-D-glucose,-D-xylosyl-(16)-rß-D-glucosyl-(14)-D-glucose and cellobiose. ¹ Present address: Department of Botany, Iowa State University,Ames, Iowa 50011, U.S.A. (Received August 29, 1981; Accepted January 12, 1982)     

Characterization of the Hydrolytic Activity of Avocado Cellulase

The cellulase produced by ripening avocado fruits (Persea americanaMill cv. Fuerte) was isolated and purified using chromatofocusing(pH 7–4) and gel filtration on a Bio- Gel P-100 column.Characteristics of the cellulase were assessed by using, assubstrates, a range of polysaccharides containing various sugarresidues and varying types of linkages between the residues.Only those substrates containing (14)-ß-glucosyl linkageswere hydrolyzed by the purified enzyme. Two polysaccharidesthat were extensively hydrolyzed by the cellulase were carboxymethylcelluloseand (13),(14)-ß-D-glucans such as from Avena endospermcell walls. Characterization of the activity in the degradationof the mixed linked glucan of Avena and cellodextrins indicatedthat the enzyme has a limit recognition-hydrolytic site of four(l4)-ß linked glucose residues. It was also foundthat the enzyme could cleave only (14)-ß-linkagesthat were adjacent to other (l4)-ß-D-glucosyl linkages.Activity of the cellulase against isolated avocado fruit cellwalls indicated that the purified enzyme was incapable of appreciablysolubilizing the cellulosic components of these walls. ¹Supported in part by National Science Foundation Research GrantPCM 7818588. ²USDA-ARS, Dairy Forage Research Center, University of Wisconsin,Madison, WI 53706. ³Department of Vegetable Crops, University of California, Davis,CA 95616. (Received September 14, 1985; Accepted February 12, 1986)     

Anti-Auxin Activity of Xyloglucan Oligosaccharides: the R?le of Groups other than the Terminal {alpha}-L-Fucose Residue

The quantitatively major nonasaccharide (XG9) derived from xyloglucanby digestion with cellulase exhibits anti-auxin activity inthe pea stem segment straight-growth bioassay; the most effectiveconcentration of XG9 is c. 10^–9 M. Previous work had shownthat XG9 owes its biological activity to the presence of a terminal-L-fucopyranose residue. In order to investigate to what extentthe remainder of the XG9 molecule is essential for activity,several fucose-containing compounds were tested for their abilityto mimic the anti-auxin effect of XG9. A fucose-containing pentasaccharideof xyloglucan (XG5; probable structure FucGalXylGlcGlc) was,at 10^–8 M, about as effective an anti-auxin as 10^–9M XG9; unlike XG9, XG5 did not diminish in effectiveness at10^–7 M. The human milk trisaccharide, 2'-fucosyl-lactose[L-fucopyranosyl--(12)-D-galactopyranosyl-ß-(14)-D-glucose],whose FucGal unit is identical with that of XG9, inhibited auxin-inducedelongation over a wide range of concentrations centred on about10^–8 M. 2'-Fucosyl-lactose at 10^–8 M was about aseffective an anti-auxin as 10^–9 M XG9. Free L-fucose andmethyl--L-fucopyranoside were unable to inhibit auxin-inducedgrowth at any concentration tested (10^–10 M to 10^–6M) and neither compound interfered with the inhibition causedby 10^–9 M XG9 when co-incubated at concentrations up to10^–4 M. The results confirm the essential r?le of an -linkedterminal fucose residue in the anti-auxin activity of XG9 andshow that the sub-terminal galactose residue may also be required.Possible reasons why high concentrations of XG9 fail to antagonizeauxin-induced growth while high concentrations of XG5 and 2'-fucosyl-lactosecontinue to do so are discussed. Key words: Anti-auxin, oligosaccharin, fucose     

Flexibility in the donor substrate specificity of {beta} 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates

Biosynthetically, bovine N-acetylglucosainine ß 1,4-galacto-syltransferase(GalT) catalyses the transfer of galactosyl residues from UDP-Galto the 4-position of GlcNAc units, resulting in the productionof N-acetyllactosamine sequences. UDP-Glc and UDP-GalNAc werealso found to act as donors for this enzyme, allowing the preparationof ßGlc(14)-ßGlcNAc and ßGalNAc(14)ßGlcNActerminating structures on the milligram scale. GalT could thusbe used to add ßGalNAc to ßGlcNAc(12)Manterminating structures, converting them to the ßGalNAc(14)ßGlcNAc(12)Mansequences found on glycoprotein hormones. GalT did not transferGlcNAc residues from UDP-GlcNAc, but it could utilize UDP-GlcNH₂as a donor. Synthesis of ßGlcNAc(14)ßGlcNAcsequences could therefore be accomplished by transfer of GlcNH₂from its UDP derivative, followed by N-acetylation of the productamino-disaccharide using acetic anhydride in methanol. The productsof the enzymatic reactions were characterized by ¹H-NMR-spectroscopyand fast-atom bombardment mass spectrometry. This work expandsthe scope of the combined chemical-enzymatic synthesis of complexcarbohydrates, using glycosyltrans-ferases, to the productionof oligosaccharides different from those for which these enzymeswere designed. These unnatural reactions should find applicationin glycoprotein and glycolipid remodelling. galactosyltransferase chemica1-enzymatic synthesis of oligosaccharides oligosaccharide analogues sugar-nucleotide analogues carbohydrate remodelling     

A novel monoantennary complex-type sugar chain found in octopus rhodopsin: occurrence of the Gal{beta}1->4Fuc group linked to the proximal N-acetylglucosaniine residue of the trimannosyl core

The N-linked sugar chains were liberated as oligosaccha-ridesfrom octopus rhodopsin by hydrazinolysis. Most of the oligosaccharideswere neutral, and separated into two major components by columnchromatography using immobilized lectins and Bio-Gel P-4. Structuralanalysis of the one major component by sequential exoglycosidasedigestion, chemical fragmentation in combination with meth-ylationanalysis revealed that it is a nonasaccharide; Man16(Gaiβ13GlcNAcβ12Man13)Manβ14GlcNAcβ14(Galβ14Fuc16)GlcNAcThis structure is quite unique in that a novel galactosylatedfucose residue is attached to the reducing terminal N-acetyl-glucosamineresidue. galactosylated Fuc N-linked sugar chain novel structure octopus rhodopsin     

Incorporation of some possible radioactive intermediates into chlorogenic acid in sliced sweet potato tissue

t-Cinnamic acid-2-¹⁴C, p-coumaric acid-2-¹⁴C and caffeic acid-2-¹⁴Cwere administered to discs of sweet potato roots and incorporationof each radioactive compound into chlorogenic acid was compared.The data suggest that chlorogenic acid is synthesized througheither or both of two major pathways, phenylalanine t-cinnamate t-cinnamoyl derivative p-coumaroyl derivative chlorogenicacid and phenylalanine t-cinnamate p-coumarate p-coumaroylderivative chlorogenic acid. ¹Part 75 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address : Department of Biology, Tokyo MetropolitanUniversity, Setagaya-ku, Tokyo. (Received December 23, 1968; )     

Human Lewis {alpha}1->3/4fucosyltransferase: specificity of fucose transfer to GlcNAc{beta}1->3Gal{beta}1->4Glc{beta}1->1Cer (LcOse3Cer)

Biosynthesis of the Le^x series of carbohydrate antigens proceedsby fucose transfer in 13-linkage to the penultimate GlcNAc residueof a neolacto-series oligosaccharide acceptor, a reaction catalysedby multiple enzymes expressed in human tissues. Particularlybroad acceptor specificity, including the ability to catalysefucose transfer to both lacto- and neolacto-series acceptorsas well as the precursor Lc₃ structure (where Lc₃ lactotriaosylceramide,is GlcNAcß13Galß14Glcß1Cer), existsfor one human fucosyltransferase form, the Lewis 13/4fucosyltransferase(FucT-III). To determine if fucose transfer to Lc₃may representan alternate early step in Le^xor Le^a antigen biosynthesis withthis enzyme, the chemical structure of the fucosylated Lc₃ reactionproduct formed by the Lewis 13/4fucosyltransferase from Colo205 cells has been defined. Transfer of [¹⁴C]fucose to Lc₃ yieldeda labelled product migrating as a tetrasaccharide on thin layerchromatography plates. This product remained an acceptor forboth ß13- and ß14-galactosyl transfer onthe terminal GlcNAc residue. The product was degraded to a fucosylatedtrisaccharide derivative by bovine kidney ß-N-acetylglucosaminidase.Fast atom bombardment mass spectrometry and methylation analysisconfirmed that the product was composed exclusively of the followingstructure containing a fucose linked to the 3-position of theinternal Glc residue: GlcNAcß13Galß14Glcß11Cer Such a structure does not represent an intermdiate in Le^xorLe^a antigen biosynthesis. Thus, the evidence suggests that Le^xor Le^a antigen synthesis results exclusively from fucosylationof complete core chains. fucosyltransferase lacto-series LcOse₃Cer Lewis antigen transfer specificity     

Further characterization of the binding properties of a GalNAc specific lectin from Codium fragile subspecies tomentosoides

Previous study on the binding properties of a lectin isolatedfrom Codium fragile subspecies tomentosoides (CFT) indicatesthat this lectin recognizes the GalNAc1 sequence at both reducingand nonreducing ends. In this study, the carbohydrate specificityof CFT was further characterized by quantitative precipitin(QPA) and inhibition of lectin-enzyme binding assays. Of theglycoforms tested for QPA, all asialo-GalNAc1 containing glyco-proteinsreacted well with the lectin. Asialo hamster and ovine submandibularglycoproteins, which contain almost exclusively Tn (GalNAclSer/Thr)residues as carbohydrate side chains, and Streptococcus typeC polysaccharide completely precipitated the lectin added, whilethe GalNAcβcontaining Tamm-Horsfall Sd(a⁺) glycopro-teinand its asialo product were inactive. Among the oligo-saccharidestested for inhibiting lectin-glycoprotein interaction, GalNAc13GalNAcβ13Gal14Galβ14GIc(Fp)and Galβ13GalNAc1benzyl (T) were the best, and about 125-foldmore active than GalNAc They were about 3.3, 6.6, and 43 timesmore active than Tn containing glycopeptides, GalNAc13(LFuc12)Gal(A_h) and Galβ13GalNAc(T), respectively. From the presentand previous results, it is concluded that the combining siteof CFT is probably of a groove type that recognizes from GalNAclto pentasaccharide(Fp). The carbohydrate specificity of thislectin can be constructed and summarized in decreasing orderby lectin determinants as follows: Fp and T > Tn cluster> A_h >>I/II. carbohydrate specificities Codium fragile tomentosoides glycoprotein binding lectins     

Effects of illumination on absorption peak shifts in spectra of intact etiolated cotyledons of Pharbitis nil I. Existence of two kinds of shift patterns

Two patterns were found in the shifts of absorption peaks inspectra of intact etiolated Pharbitis cotyledons illuminatedat room temperature. One was a well-known pattern, P649C678C683C672,called the "high-intensity illumination pattern" in this study.The other, called the "low-intensity illumination pattern,"was P649C672. (Received June 16, 1976; )     

Comparative Biochemistry of the Pineal Gland

The pineal gland is biochemically very active. In mammals, ithas the unique capacity to synthesize the hormone melatonin(N-acetyl-5-methoxy-tryptamine). Although the synthesis of melatoninis confined mainly to the pineal gland of all vertebrates, theeyes and brains of amphibians and fish also can form melatonin.Melatonin is synthesized in the pineal as follows: tryptophan 5-hydroxy-tryptophan serotonin N-acetylserotonin melatonin.The final step is catalyzed by the enzyme, hydroxyindole-O-methyltransferase (HIOMT), which is highly localized in the pinealof all vertebrate species examined. The activity of HIOMT ischanged when animals are kept in constant darkness or light.In rats, highest HIOMT activity is present in constant darkness,while the reverse occurs in avian species. In mammals, informationabout lighting reaches the pineal via the retina inferior accessoryoptic tract preganglionic sympathetic fibers superior cervicalganglia postganglionic fibers pineal parenchymal cells. Lightingmessages reach the hen's pineal via a nonretinal pathway. Studieswith tissue culture indicate that noradrenaline liberated fromsympathetic nerves stimulates synthesis of melatonin. Thereare circadian rhythms in pineal serotonin content which areendogenous and abolished by removal of superior cervical gangliaor by decentralization. There is also a 24-hour rhythm in pinealnoradrenaline. This rhythm is exogenous and is abolished byblinding or cutting the inferior accessory optic tract.     

The Effects of Salinity upon Galactosyl-Glycerol Content and Concentration of the Marine Red Alga Porphyra purpurea (Roth) C.Ag.

Changes in the major alcohol-soluble, low molecular weight carbohydratesof P. purpurea, O--D-galactopyranosyl-(1-2)-glycerol (‘floridoside’)and O--D-galactopyranosyl-(1-1)-glycerol (‘isofloridoside’),have been examined in response to salinity variation. ‘Floridoside’is shown to vary in absolute amount, increasing in hypersalineand decreasing in hyposaline media. ‘Isofloridoside’content per cell does not change in a similar manner. Responsesare almost identical under light or dark conditions, ‘floridoside’changes being complete within 24 h. Decreasing the externalwater potential using ionic and non-ionic solutes has the sameeffect upon galactosyl-glycerol content. The amount of ‘floridoside’synthesized, and degraded under hypersaline and hyposaline conditionsrespectively is shown to be insufficient to restore cell volumeto its original value. It is therefore suggested that the primaryfunction of ‘floridoside’ increases in concentratedsea-waters is that of a compatible solute, serving to protectthe cell during periods when the external salt content is increaseddramatically.     

Chlorophyll Formation in the YG-6 Mutant of Chlorella regularis: Spectral Characterization of Protochlorophyllide Phototransformation

Dark-grown YG-6 mutant cells of Chlorella regularis accumulateat least two forms of phototransformable protochlorophyllide(Pchlide) with in vivo absorption maxima at 634 nm (Pchlide634) and 650 nm (Pchlide 650). Difference spectrophotometricanalyses and the action spectra showed that Pchlide 634 is firsttransformed into the 648 nm form and then phototransformed intochlorophyllide (Chlide) 672 nm. Pchlide 650 is phototransformedinto Chlide 685 which then shifts towards short wavelength-formingChlide 667 in the subsequent dark stage (Shibata shift). Pchlide650 is regenerated at the expense of photoinactive Pchlide 632.In washed cells after the phototransformation, the Shibata shiftwas accelerated. Freezing/thawing treatment in the dark causedconversion of phototransformable Pchlide 650 into photoinactivePchlide 633, but phototransformation activity of Pchlide 634still partly remained. These results suggest that in the final step of light-dependentchlorophyll formation in the YG-6 mutant of C. regularis, twosequentially and functionally separate routes are present: (1) Pchlide 634 Pchlide 648 Chlide 672 Chlorophyll a. (2) Pchlide 650 Chlide 685 Chlide 667 Chlorophyll a. (Received June 4, 1983; Accepted November 11, 1983)     

Separation of partially desialylated branched oligosaccharide isomers containing {alpha}(2<-3)- and {alpha}(2<-6)-linked Neu5Ac

The following two tri-sialylated triantennary oligosaccharides,which differ only in the linkage of the Neu5Ac to the uppermostbranch were, individually, partially desialylated to produceall possible di- and mono-sialylated isomers. A tetra-sialylated triantennary isomer, which contained an (26)-linkedNeu5Ac to the GlcNAc on branch III, was also converted to allpossible trisialylated isomers by mild acid hydrolysis as previouslydescribed (Roher et al., Anal Biochem., 212, 7–16, 1993).The resulting branch isomers were separated using high-pH anion-exchangechromatography (HPAEC). Structures were assigned to peak fractionson the basis of the previously described effect of (26)- and(23)-linked Neu5Ac on the elution order of branched lactosamine-typeoligosaccharides (Townsend et al., Anal Biochem., 182, 1–8,1989). No differences in the acid lability of the Neu5Ac linkageto either Gal ((23) or (26)) or GlcNAc ((26)) were observed.Our studies show that chemical desialylation and HPAEC is auseful approach to prepare and identify all possible sialylatedbranch isomers and should prove useful for defining the branchspecificity of sialyltransferases and sialidases. high-pH anion-exchange chromatography pulsed electrochemical detection sialidases sialylated oligosaccharides sialyltransferases     

AGMATINE AND N-CARBAMYLPUTRESCINE AS INTERMEDIATES IN THE FORMATION OF NICOTINE BY TOBACCO PLANTS

Agmatine-G-³H and N-carbamylputrescine-l,4-¹⁴C were effectivelyincorporated into the nicotine of tobacco plants. This resultmay indicate a route that the pyrrolidine ring of nicotine isformed from putrescine by the following pathway: arginineagmatineN-carbamylputrescineputrescinepyrrolidinering. (Received February 7, 1966; )     

Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay

Information on the biosynthesis of the D-arabinans of the cellwall of Mycobacterium tuberculosis is rapidly emerging, withthe promise of new targets for drug development against tuberculosis.Accordingly, arabinosyl transferase assays were developed utilizingsynthesized [1–¹⁴C]-β-D-arabinofuranosyl-1-monophosphoryldecaprenolas donor and a variety of O- and S-alkyl arabinosides as acceptors.These were: -D-Araf-(15)--D-Araf-O- and -S-alkyl di-arabinosidesand -D-Araf-(15)--D-Araf-(15)--D-Araf-O- and -S-alkyl triarabinosides.Whereas the O- and S-alkyl monosaccharide acceptors were inactive,the O- and S-alkyl disaccharide and the O- and S-alkyl trisaccharideacceptors (<C12) possessed considerable acceptor activity,and the trisaccharide acceptors were more potent than the correspondingdisaccharides. The O-alkyl disaccharide acceptors with a C8alkyl chain were more active than those containing the C6 orC10 analogs. Chemical analysis of the enzymatically synthesizedproducts of the reactions demonstrated that β-D-arabinofuranosyl-1-monophosphoryldecaprenolwas an effective donor for two of the three potential arabinosyltransferases: β-D-arabinofuranosyl-1-monophosphoryldecaprenol:arabinan (15) arabinosyl transferase and β-D-arabinofuranosyl-1-monophosphoryl-decaprenol:arabinan β(12) arabinosyl transferase. The β(12) arabinosyltransferase activity was more in evidence in the presence ofthe O-alkyl disaccharide acceptor, whereas both transferaseswere about equivalent in the presence of the S-alkyl trisaccharideacceptor. The tuberculosis drug, ethambutol, a known mycobacterialarabinosyl transferase inhibitor, was inactive within thesearabinosyl transferase/acceptor based assay systems, supportingother evidence that a third activity, responsible for the formationof 13 linkage, is the drug target. acceptor arabinan biosynthesis glycosyltrans-ferase assay mycobacteria     

Long chain fatty acid synthesis in developing castor bean seeds II. Operation of the path from sucrose to long chain fatty acids in a subcellular particulate system

Particles spun down at 10,000 ? g from developing castor beanseeds were capable of synthesizing LFAs from sucrose, a physiologicalprecursor transferred from leaves as a photosynthetic product.Tracer experiments, in combination with inhibitor effects, intermediatedilutions and cofactor requirements, indicated the operationof the following path: sucroseUDPGG-1-PG-6-PGAPpyruvateacetyl-CoAmalonyl-CoALFA.The whole path appears to be associated with 10,000 ? g particles,because repeated washings were unsuccessful in dissociatinga partial path from the particles, depsite of disorganizingthe structure of the particles. Based on the occurrence of freehexose(s) and the utilization of UDPG similar to that of sucroseor G-1-P in this reaction, it is probable that hexose phosphateis formed from sucrose via UDPG and fructose, although the conversionof sucrose to hexose phosphates via glucose and furctose isnot ruled out. Inhibitor experiments showed that ATP is self-supportingover the whole path, the ATP formed in the glycolytic path beingconsumed in a acetyl-CoA carboxylation step. Since oxidizedpyridine nucleotides are as available as reduced ones for LFAsynthesis, they seem to shuttle between the reduction in theconversion of sucrose to acetyl-CoA and the oxidation in LFAsynthesis from acetyl-CoA. From the pattern of the LFAs synthesized,NAD⁺ is available for the synthesis of saturated LFAs (18: 0,16: 0). whereas NADP⁺ is available for that of unsaturated LFAs(18: 1, 16: 1). (Received July 23, 1973; )     

Concanavalin A Inhibits Auxin-Induced Elongation and Breakdown of (1 -> 3), (1 -> 4)-{beta}-D-Glucans in Segments of Rice Coleoptiles

Concanavalin A (Con A) suppresses auxin-induced elongation ofsurface-abraded segments from both dicotyledonous and poaceousplants. In coleoptile segments of rice (Oryza sativa L.), theauxin-induced decrease in the minimum stress-relaxation timeand increase in the mechanical extensibility of the cell wallswere also inhibited by Con A, indicating that the lectin suppresseselongation by inhibiting the cell wall loosening. Auxin causeda decrease in the level of (1 3), (1 4)-ß-D-glucansin the cell walls of rice coleoptile segments, and this decreasewas also inhibited by the lectin. Con A suppressed the autolytichydrolysis of the glucans, as well as their breakdown in vitroby a protein fraction that had been extracted from the cellwalls of rice coleoptiles with 1 M NaCl. Furthermore, most ofthe glucan-hydrolyzing activity of the wall proteins bound toa Con A-Sepharose column, suggesting that glycoprotein enzymesare involved in the hydrolysis. Although Con A also affectedthe hydrolysis of other wall polysaccharides, the present data,when considered in combination with the inhibitory effects ofglucan-specific or glucanasespecific antibodies, support theview that the breakdown of (1 3),(1 4)-ß-D-glucansis associated with the cell wall loosening that is responsiblefor auxin-induced elongation in Poaceae. (Received August 17, 1994; Accepted February 15, 1995)     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasinglyimplicated as regulators of membrane signaling events. Macrophageganglioside patterns dramatically increase in complexity whenmurine peritoneal macrophages are stimulated in vivo with theappearance of the sialidase-sensitive monosialoganglioside GMlb(cisGMl) as a major component Gangliosides from stimulated murineperitoneal macrophages were separated into monosialo and polysialofractions and the polysialo fraction structurally characterizedby enzymatic, chemical, and mass spectra methods. All detectablecomponents of the polysialo fraction were determined to be disialogangliosides.Treatment of the polysialo fraction with Clostridium perfringenssiali-dase produced mostly the sialidase-resistant monosialoganglioside,GMIa, and a minor amount of asiaJoGMI. Perio-date oxidationand mass spectrometry analyses demonstrated the lack of tandemdisialo moieties which indicated the absence of GD1b or GD1c(GDI) entities. The combined data showed the major disialogangliosidesconsisted of GDla entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer,IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, andIV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted ofGDl entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGcGgOse4Cer,and also positional iso-mer(s) of GDl(NeuAc, NeuGc). These isomericcomponents were identified by collision analysis and tandemmass spectrometry. Consistent with previous analyses, the cer-amideportion of all polysialo (disialo) gangliosides contained solelyC18 sphingosine with C16 and C24 fatty acid moieties. Theseresults, combined with the previous characterization of macrophagemonosialogangliosides, indicate normal murine macrophage gangliosidebiosynthesis proceeds along the "a" ganglioside pathway, e.g.,GM3GM2GMlaGDl, and the proposed asialogan-glioside or "" pathway,asialoGMlGMlbGDl. The presence of totally sialidase-sensitivegangliosides appears to be characteristic of functional murineperitoneal macrophages while they are reduced in geneticallyimpaired cells. ganglioside GDla ganglioside GDl murine macrophages tandem mass spectrometry collision induced dis-association electrospray ionization