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     

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     

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     

Hydrophobic mannosides act as acceptors for trypanosome {alpha}-mannosyltransferases

A series of hydrophobic mannosides were synthesized and testedfor their ability to act as acceptor substrates for mannosyltransferasesin a Trypanosoma brucei cell-free system. The thiooctyl -mannosidesand octyl -mannosides all accepted single mannose residues in-linkage, as judged by thin layer chromatography of the productsbefore and after jack bean -mannosidase digestion. The mannosylationreactions were inhibited by amphomycin, suggesting that theimmediate donor was dolicholphosphate-mannose (Dol-P-Man) inall cases. The transferred -mannose residues were shown to beboth 1-2 and 1-6 linked by Aspergillus phoenicis -mannosidaseand acetolysis treatments, respectively. These data suggestthat the compounds can act as acceptor substrates for the Dol-P-Mandependent 1-2 and 1-6 mannosyltransferases of the GPI biosyntheticpathway and/or the dolichol-cycle of protein N-glycosylation.One of the compounds, Man1-6Man1-O-(CH₂)₇CH₃, inhibited endogenousGPI biosynthesis in the cell-free system, suggesting that itcould be a substrate for the trypanosome Dol-P-Man:Man₂GlcN-Pl1-2 mannosyltransferase. dolichol glycosylphosphatidylinositol mannosyltransferase trypanosome     

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     

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle {psi} for the orientation of {alpha}1,6-arm

The conformation of the heptasaccharide Man-1,6-(Man-1,3)(Xyl-ß1,2)-Man-ß,4-GlcNAc₂-ß1,4-(L-Fuc-1,3)-GlcNAc₁,the carbohydrate moiety of Erythrina corallodendron lectin (EcorL),the hexasaccharide Man-1,6-(Man-1,3) (GlcNAc-ß1,4)-Man-ß1,4-GlcNAc-ß1,4-GlcNAcand their disaccharide fragments have been studied by moleculardynamics (MD) simulations for 1000 ps with different initialconformations. In the isolated heptasaccharide, the most frequentlyaccessed conformation during MD has a value of 180° aroundMan-1,6-Man linkage. This conformation is stabilized by theformation of a hydrogen bond between the carbonyl oxygen ofGlcNAc₂ with the O3/O4 hydroxyls of the 1,6-linked mannose residue.The conformation of the heptasaccharide found in the crystalstructure of the EcorL-lactose complex (Shaanan et al., Science,254, 862, 1991), that has a value of 76° around Man-1,6-Manlinkage, is accessed, although less frequently, during MD ofthe isolated oligosaccharide. The ,, = 58°,–134°,–60°conformation around Man-₁,6-Man fragment observed in the crystalstructure of the Lathyrus ochnrs lectin complexed with a biantennaryoctasaccharide (Table I in Homans,S.W., Glycobiology, 3, 551,1993) has also been accessed in the present MD simulations.These values for the 1,6-linkage, which are observed in theprotein-carbohydrate crystal structures and are accessed inthe MD simulations, though occasionally, have not been predictedfrom NMR studies. Furthermore, these different values of leadto significantly different orientations of the 1,6-arm for thesame value of . This contrasts with the earlier predictionsthat only different values of can bring about significant changesin the orientation of the ₁,6-arm. The MD simulations also showthat the effects of bisecting GlcNAc or ß1,2-xyloseare very similar on the ₁,3-arm and slightly different on the₁,6-arm. bisecting GlcNAc carbohydrates glycoprotein lectinsaccharide complex     

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     

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     

Phosphorylation and subcellular location of {alpha}-L-fucosidase in Iymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

Mannose 6-phosphate is a recognition marker used by many newlymade acid hydrolases for their transport to lyso-somes. Previously,we investigated the incorporation of ³²Pi into -L-fucosidaseof lymphoid cell lines from a healthy individual (control) andan I-cell disease patient [DiCioccio and Miller, Glycobiology,1, 595–604 (1991)]. Phosphoserine was found in immunoprecipitable-L-fucosidase of both control and I-cell lymphoid cells, butmannose 6-phosphate was identified only in enzyme of controlcells. Extension of this investigation to lymphoid culturesof a pseudo-Hurler polydystrophy patient also identified onlyphosphoserine in -L-fucosidase. Using [³H] mannose instead of³²Pi, the precise identification of mannose 6-phosphate in -L-fucosidaseof control cells, and its absence in -L-fucosidase of I-celland pseudo-Hurler cells, was established. The stoichiometryof phosphorylation of -L-fucosidase in I-cell, pseudo-Hurlerand control lymphoid cells was 3, 4 and 10 mol Pi/mol enzyme,respectively. -L-Fucosidase was located in lysosomes isolatedfrom control, I-cell and pseudo-Hurler lymphoid cells by subcelluarfractionation on Percoll density gradients. Both I-cell andpseudo-Hurler lymphoid cells displayed normal intralysosomalactivity of -L-fucosidase despite lack of the mannose 6-phosphatemarker. Thus, I-cell and pseudo-Hurler lymphoid cells must possessa mannose 6-phosphate-independent mechanism for directing -L-fucosidaseto lysosomes. Phosphorylation of -L-fucosidase in pseudo-Hurlerand I-cell lymphoid cultures was found almost exclusively inintracellular and not in extracellular enzyme, suggesting thatphosphoserine may participate in the localization of -L-fucosidasein lysosomes of these cells. -L-fucosidase I-cell disease lysosome phosphorylation pseudo-Hurler polydystrophy     

Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi

In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi     

A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

The 1,2-fucosyltransferase family (1,2FT) is the largest familyof glycosyltransferases in the genome of the free-living nematodeCaenorhabditis elegans, and early evidence suggests that eachmember may have a unique activity. Here we describe a C. elegansgene (designated CE2FT-2) encoding an 1,2FT that has the potentialto generate the sequence Fuc1-2Galβ1-3GalNAc-R, which isthe H-type 3 blood group structure. The CE2FT-2 cDNA encodesa putative transmembrane protein that shows 42% amino acid identityto a previously cloned C. elegans 1,2FT (termed CE2FT-1), buthas a very low identity (16–20%) to 1,2FT sequences inhumans, rabbits, and mice. A recombinant form of CE2FT-2 expressedin human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP,but unexpectedly, the enzyme is inactive toward the acceptorGalβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTspreviously described from animals that all utilize Galβ-O-phenyl.CE2FT-2 is expressed at all stages of worm development, butremarkably, promoter analysis of the CE2FT-2 gene using greenfluorescent protein reporter constructs indicates that the CE2FT-2is expressed exclusively in pharyngeal cells of the worm fromembryo to an adult stage. Because pharyngeal cells are knownto secrete their glycoconjugates to the nematode surface, theseresults may indicate that products of CE2FT-2 contribute tointeractions of the nematode with its environment or are usedas ligands for bacterial attachment. These findings, along withthose on other 1,2FTs in C. elegans, suggest that each 1,2FTin this organism may have a unique acceptor specificity, expressionpattern, and biological function.     

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     

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)     

Sialylation of n-alkyllactosides, galactosides and glucosides by sialyltransferases from rat liver Golgi vesicles

nAlkyl - and -lactosides, galactosides and glucosides with differentalkyl chain lengths (C₂, C₈, C₁₄, and C₂₀) were synthesizedand used as acceptors for sialyltransferases from rat liverGolgi vesicles. The -galactosides, -glucosides, and both - and-lactosides, were sialylated. Keeping the acceptor concentrationconstant, sialylation rates reached a maximum for the n-octyl- and -lactosides, n-Octyl -galactoside and noctyl -glucoside,respectively. noctyl -glucoside, respectivwly. n-Octyl -galactosideand n-octyl -glucoside were not sialylated. The reaction productswere characterized by TLC. With n-octyl lactoside and galactosideas acceptors, two major sialylation products were formed. Thjeycould be separated by preparative TLC, and their structureswere identified as 2–3 and 2–6 sialylated acceptors,respectively, by a combination of periodated oxidation, NaBD₄reduction,permethylation and subsequent analysis by fast atombombardment mass spectrometry (FAB-MS). The structure of thesingle product obtained from n-ictyl -glucoside was determinedto be the 2–6 sialylated glucoside. Competition experimentswith n-octyl lactoside and lactosylceramide and gangliosideGal1-3GalNAc1-4(NeuAc2–3)Gal1–4Glcbeeta1–1Cer(GM1) as acceptors for sialyltransferases suggested that SAT-I[NeuAc2–3Gal1–4Glc1-1Cer (GM3) synthase] was atleast in least in part responsible for the 2–3 sialylationof n-octyl lactoside. alkylgalactosides alkylglucosides alkyllactosides neoglycolipids sialytransferases     

Biosynthesis, processing, and secretion of {alpha}-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

N-Acetylglucosamine 1-phosphotransferase is a key enzyme requiredfor synthesis of the mannose 6-phosphate recognition markerthat is used by many newly made acid hydrolases for their transportto lysosomes. It has previously been found that lymphoid cellsfrom patients with I-cell disease and pseudo-Hurler polydystrophyhave nearly normal intracellular and intralysosomal activitiesof several lysosomal acid hydrolases, despite a deficiency ofN-acetylglucosamine 1-phosphotransferase. These results suggestthat lymphoid cells may provide an important system to investigatealternate mechanisms for targeting newly made acid hydrolasesto lysosomes. In the present study, the biosynthesis, processingand secretion of -L-fucosidase in I-cell and pseudoHurler lymphoidcells was used as a model system to study the existence of suchmechanisms. The level of intracellular -L-fucosidase proteinin exponentially growing I-cell or pseudo-Hurler lymphoid cultureswas statistically indistinguishable from the mean of 19 controlcultures. A 1.5 h [³⁵S]methionine pulse experiment showed that-L-fucosidase is initially sythesized by I-cell, pseudo-Hurlerand control cultures as an intracellular form (M_r = 58 000).Companion cultures chased with methionine from 2 to 21 h processedthe enzyme to an intracellular form (M_r = 60 000) and an extracellularform (M_r = 62 000). All enzyme forms were glycoproteins withpolypeptide chains of Mr 52 000. In control cells incubatedwith radioactive inorganic phosphate (³²Pi), <1% of the ³²Piincorporated into -L-fucosidase was associated with carbohydratechains and >99% with polypeptide chains. In I-cell diseaselymphoid cells, the ³²Pi incorporated into -L-fucosidase wasassociated solely with polypeptide chains. A qualitative analysisof phosphorylated residues identified phosphoserine in -L-fucosidasefrom control and I-cell lymphoid cells. Only -L-fucosidase fromcontrol cells contained mannose 6-phosphate. These results areconsistent with the proposal that I-cell lymphoid cells mayuse a mannose 6-phosphate-independent mechanism for routing-L-fucosidase. Additional metabolic labelling experiments demonstratedthe presence of ³²P-labelled -L-fucosidase in both cells andmedium of a control lymphoid culture, but only in cells of anI-cell lymphoid culture. In contrast, -L-fucosidase labelledwith [³⁵S]methionine was found in cells and medium of controland I-cell lymphoid cultures. Since phosphoserine was only foundto occur in intracellular, but not in extracellular -L-fucosidaseof the I-cell culture, we speculate that phosphoserine may beinvolved in intracellular retention of -L-fucosidase in I-celllymphoid cells. -L-fucosidase I-cell disease lymphoid cells phos-phorylation pseudo-Hurler polydystrophy     

Hearing and glycoconjugates: localization of Ley, Lex and sialosyl-Lex in guinea pig cochlea, particularly at the tectorial membrane and sensory epithelia of the organ of Corti

Immunohistological examination of guinea pig cochleas was performedusing a panel of 25 monoclonal antibodies directed to variouslacto-, ganglio- and globo-series carbohydrate epitopes as wellas mucin-type epitopes. Lacto-series structures were found tobe localized at specific sites of the tectorial membrane (TM)and Corti's organ, i.e. 13 fucosyl type 2 chain (Le^x) at Kimura'smembrane, marginal band and covering net of TM; 12, 13 difucosyltype 2 chain (Le^y) at covering net; and sialosyl-Le^x and sialosyl-iat Kimura's membrane and sensory epithelia, particularly sensorytips of hair cells of Corti's organ. In striking contrast, ganglio-seriesstructures (GM3, GD3, GD2, 9-O-Ac-GD3) were detected at spiralganglion cells, neuronal fibres and stria vascularis, but werecompletely absent from Corti's organ and most of the TM. Otherepitope structures defined by various antibodies were not detectableat any location. The functional roles of lacto-series carbohydrateepitopes expressed at TM and Corti's organ remain unknown. However,the expression of Le^y (but not other structures) in associationwith developmental deficiency of TM induced by 6-N-propyI-2-thio-uracilin rats suggests that Le^y plays some role in normal TM development.The presence of Le^x at Kimura's membrane and sialosyl-Le^x athair cell sensory tips of Corti's organ suggests the intriguingpossibility that these fucosylated/sialosylated carbohydratestructures play some role in interactions (either attractiveor repulsive) of these inner ear components, which have beenimplicated in the physiology of hearing, i.e. the conversionof sound waves to nerve impulses. cochlea Corti's organ glycoconjugate sialosyl fucosyl type 2 chain tectorial membrane     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

A Transient Stimulation of Net Photosynthesis of Rye Leaves by {alpha}-Hydroxy-2-pyridinemethanesulphonic Acid ({alpha}-HPMS) due to Inhibition of Photorespiratory CO2 Release

The effects of -hydroxy-2-pyridinemethanesulphonic acid (-HPMS)upon net photosynthesis (P_n, the CO₂ compensation point (),post-lower illumination burst of CO₂ (PLIB) and post-lower temperatureburst of CO₂ (PLTB) in detached rye (Secale cereale L.) leaveswere investigated. At low concentrations ( 0.5 mol m^–3),-HPMS initially stimulated P_n and decreased the magnitude ofboth PLIB and PLTB. The decreased at all concentrations of-HPMS (0.05–5.0 mol m^–3. The effects of -HPMS onP_n and were time-dependent and, after a few minutes, the P_nwas inhibited while values increased considerably. At a higherconcentration (5.0 mol m ^–3), the transient effects of-HPMS were shorter () or not observed at all (P_n. Both PLIBand PLTB, when expressed in relation to P_n, increased at higherlevels of this compound. Similar data with respect to the effectsof -HPMS on PLIB and PLTB were found for leaves of dandelion(Taraxacum officinale L.). The results suggest that -HPMS may stimulate P_n by inhibitingphotorespiration, as originally suggested by Zelitch (1966),but only at low concentrations and over a short time span. Thedecrease of PLIB and PLTB values at low -HPMS levels is consistentwith these processes being a residual activity of the glycolatepathway. Key words: CO₂ compensation point, -hydroxy-2-pyridinemethanesulphonic acid, photorespiration, photosynthesis