Synthesis ofN-Acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy--d-glucopyranoside and 2-acetamido-2-deoxy--d-xylopyranoside, two allyl 2-acetamido-2-deoxy--d-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy--d-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific forN-acetylglucosamine. We found that: 1) The inhibitory potency ofN-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3-or 4-OH group ofN-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.Abbreviations XyLNAc N-acetyl-d-xylosamine - BSA bovine serum albumin - NeuAc N-acetylneuraminic acid - GlcNAc34-BSA amidino-type neoglycoprotein [6] containing on the average 34N-acetylglucosaminyl residues per BSA molecule     

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence,spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-GalNAc and for jack fruit agglutinin (JFA) is Me-Gal. Examination of the percentage enhancement and association constants (1.51×10⁶, 6.56×10⁶ and 4.17×10⁵ M^–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×10⁵, 1.3×10⁴, and 11.7×10⁵ M^–1 sec^–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10^–2, 83.3 sec^–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.Abbreviations SBA Soybean agglutinin - WBA I Winged bean agglutinin (Basic) - JFA Jack fruit agglutinin - PNA Peanut agglutinin - Con A Concanavalin A - Dansyl (Dns) 5-dimethylaminonaphthalene-I-sulphonyl - 2GaINDns N-dansylgalactosamine - dGal 2-deoxygalactose - l-Ara l-arabinose - d-Fuc d-fucose - l-Rha l-rhamnose - N-acetyllactosamine Galß4GlcNAc - melibiose Gal6Glc     

Lectin-binding studies of a human skin mastocytoma

Summary A solitary mastocytoma of the skin was investigated to assess the lectin-binding pattern of human mast cells. Of 18 Fluorescein-labelled lectins tested, nine reacted with mast cell granules. While lectins recognizingN-acetylgalactosamine or fucose residues did not stain mast cells, lectins with binding sites forN-acetylglucosamine, -methyl mannopyranoside, galactose, complex carbohydrates ofN-acetyl-lactosamine type and sialic acid gave a positive reaction.     

Characterization of the sugar-binding specificity of the toxic lectins isolated from Abrus pulchellus seeds

The sugar-binding specificity of the toxic lectins from Abrus pulchellus seeds was investigated by combination of affinity chromatography of glycopeptides and oligosaccharides of well-defined structures on a lectin-Sepharose column and measurement of the kinetic interactions in real time towards immobilized glycoproteins. The lectins showed strong affinity for a series of bi- and triantennary N-acetyllactosamine type glycans. The related asialo-oligosaccharides interact more strongly with the lectins. The best recognized structures were asialo-glycopeptides from fetuin. Accordingly, the kinetic interaction with immobilized asialofetuin was by far the most pronounced. Human and bovine lactotransferrins and human serotransferrin interacted to a lesser extent. The interaction with asialofetuin was inhibited by galactose in a dose dependent manner. Lactose, N-acetyllactosamine and lacto-N-biose exhibited similar degree of inhibition while N-acetylgalactosamine was a poor inhibitor. These results suggested that the carbohydrate-binding site of the Abrus pulchellus lectins was specific for galactose and possess a remarkable affinity for the sequences lactose [-D-Gal-(14)-D-Glc], N-acetyllactosamine [-D-Gal-(14)-D-GlcNAc] and lacto-N-biose [-D-Gal-(13)-D-GlcNAc].     

The plasma membrane ofPhysarum cell fragments: a morphological and electrophysiological study

Summary Small, spherical cell fragments derived from macroplasmodia of the acellular slime moldPhysarum polycephalum by incubation in a 15 mM caffeine solution were investigated with morphological and electrophysiological techniques. Analysis of cell surface composition with the fluorescence microscope and different RITC-conjugated lectins revealed strong binding of ConA and RCA-I, weak binding of PEA, DBA and WGA and no binding of UEA-I. In addition, binding sites for external calcium ions were detected by chlorotetracycline-fluorescence. Electron microscopical staining with ruthenium red, iron or lanthanum delivered evidence for localization of lectin and calcium binding sites in a thin mucous layer on the cell surface.Electrical recordings by means of intracellular microelectrodes yielded an average membrane potential (MP) of –113 mV. Spontaneous depolarizations of the MP, with amplitudes between 10 and 80 mV and a duration of 20–30 s, failed to show a correlation with contractile activity. The ionic nature of MP was studied by varying the composition of the perfusing medium. The MP was not much affected by changes in external [Ca²⁺], [K⁺], or [Na⁺] but was sensitive to changes in [Cl^–] or [H⁺], with a linear dependence on pH₀ in the range between 7 and 5. Metabolic inhibition by potassium cyanide or low temperature (11°C) as well as application of the protonophore CCCP caused a depolarization of the MP. The results strongly support the hypothesis that the MP inPhysarum cell fragments is mainly generated by an electrogenic H⁺-extrusion mechanism.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - ConA Concanavalin agglutinin - CTC chlorotetracycline - DBA Dolichos biflorus agglutinin - EGTA ethylene glycol-bis(-amino ethylether) N,N,N,N-tetracetate - PBS phosphate-buffered saline - PEA Peanut agglutinin - PIPES 1,4-piperazinediethanesulfonic acid - RCA-I Ricinus communis agglutinin I - RITC rhodamine isothiocyanate - SBA Soy bean agglutinin - UEA-I Ulex europeaeus agglutinin I - WGA Wheat germ agglutinin The paper is dedicated to Prof. Dr. K. E. Wohlfarth-Bottermann on the occasion of his 65th birthday.     

Evidence from lectin-binding studies for abnormal glycosylation ofβ-hexosaminidase in the leukaemic cell-line CCRF/CEM

The lectin affinities of -N-acetyl-d-hexosaminidase (E.C.3.2.1.52) from an acute lymphoblastic leukaemic cell-line (CCRF/CEM), a non-malignant lymphoblastic cell-line (SM1) and normal human fibroblasts were studied for both mature and precursor forms of the enzyme. Four immobilised lectins concanavalin A-Sepharose wheat germ agglutinin-Agarose,Ricinus communis agglutinin I-Agarose,Phaseolus vulgaris erythroagglutinin-Agarose and a column of serotonin-Sepharose were used. The activities of -hexosaminidase from fibroblasts and SM1 cells generally behaved similarly while the CCRF/CEM enzyme exhibited different binding patterns. Differences were also noted between precursor and mature enzyme from each cell type consistent with changes in glycosylation between the precursor form and the mature form appearing in the lysosome. These results suggest that changes in the glycosylation of -hexosaminidase, and possibly other lysosomal enzymes, may be associated with malignancy.Abbreviations Con A concanavalin A-Sepharose - RCA-I Ricinus communis agglutinin I-Agarose - WGA wheat germ agglutinin-Agarose - PHA-E Phaseolus vulgaris erythroagglutinin-Agarose - SER serotonin-Sepharose: non-T - non-B ALL non-T, non-B cell acute lyphoblastic leukaemia - 4-MU-GLcNAc 4-methylumbelliferyl 2-acetamido-2-deoxy--D-glucopyranoside     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

Appearance of lectin-binding sites during vascularization of the primordium of the central nervous system in 10 to 12-day-old mouse embryos

Summary In the present study lectin-binding sites were investigated for the lectins Ricinus communis agglutinin (RCA I), wheat germ agglutinin (WGA), soya bean agglutinin (SBA), concanavalin A (Con A), Lotus tetragonolobus(LTA) and Limulus polyphemus agglutinin (LPA) during the initial stages of vasculogenesis of the CNS-anlage in 10 to 12-day-old NMRI mouse embryos. Specific binding sites for the lectins RCA I (sugar specificity: -D-galactose, N-acetylgalactosamine), WGA (sugar specificity: N-acetylglucosamine, sialic acid), and SBA (sugar specificity: N-acetylgalactosamine, -D-galactose) were detected in the newly formed capillaries within the neuroepithelial cell layer. In contrast, binding sites for Con A, LTA and LPA could not be observed at the start of the vascularization of the CNS-anlage. From these results, the conclusion can be drawn that glycoconjugates containing D-galactose, N-acetylgalactosamine and N-acetyl-glucosamine moieties are involved in the early vasculogenesis of the embryonic CNS-anlage of the mouse.     

Diabetes mellitus associated changes in glomerular glycocompounds: A fluorescence microscopical study

Summary Renal biopsy specimens showing histological alterations typical of advanced diabetic glomerulopathy were studied for changes in glomerular glycoconjugates, using fluorochrome-coupled lectins as probes. All samples studied showed a marked reduction in the binding ofTriticum vulgaris (WGA) lectin in the glomerular basement membranes. On the other hand, new glomerular binding sites for the lectins ofDolichos biflorus (DBA),Helix pomatia (HPA) andArachis hypogaea (PNA), recognizing galactosyl moieties of glycoconjugates and giving no reaction in normal glomeruli, were seen in all samples studied. In addition,Wistaria floribunda lectin (WFA), recognizing galactosyl and.N-acetylgalactosaminyl configurations in glycoconjugates, gave a typical linear binding along the glomerular basement membranes, differing markedly from its reaction with normal kidney.Ulex europaeus (UEA I) showed reduced binding in the glomeruli of diabetic nephropathy.The results show that changes in glomerular glycoconjugates may appear in diabetic nephropathy, suggesting a disturbance in the turnover of the non-reducing terminal saccharide residues. In addition, the results show that lectins are useful probes for studying these changes further.     

Monoclonal antibody raised against murine IL-1 α peptide cross-reacts with a 60-kDa antigen in early Drosophila melanogaster embryo

Whole-mounts of Drosophila embryos were stained with the monoclonal antibody Vmp 18, raised against the peptide 199–208 of murine interleukin 1/. Immunofluorescence observations showed that the antibody cross-reacted with an antigenic determinant that changed in localization during Drosophila development. In syncytial Drosophila embryos, the antibody recognized an epitope localized on the nuclear envelope throughout mitotic division. As cellularization occurred, the fluorescence was mainly concentrated in the apical region of the blastoderm cells. Western blot analysis of whole Drosophila embryo extracts showed that the antibody recognized a 60-kDa protein in syncytial embryos and during germ band elongation. This suggests that the 60-kDa antigen undergoes dynamic redistribution during embryogenesis.This work was supported in parts by grants from the Italian MURST (40% and 60% funds) and from the Consorzio Siena Ricerche     

Histochemical study of Hurler's disease by the use of peroxidase-labelled lectins

Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry

Summary Many of the flagellates inhabiting the hindgut of lower termites are associated with ectobiotic, rod-like bacteria or spirochetes. Different types of attachment sites are present. Electron dense material underlies, e.g., the plasma membrane ofJoenia annectens at the contact site, whereas other attachment sites do not show any visible specializations. The host cell's glycocalyx may, however, be reduced at the attachment sites as it is the case inDevescovina glabra. The thick glycocalyx ofStephanonympha nelumbium is not changed at the sites where bacterial rods attach, but spirochetes penetrate to a certain extent. Bacteria which colonize the extracellular surface structures ofMicrorhopalodina multinucleata express their own glycocalyx to mediate a contact. In this study we focussed on the examination of one common mode of interaction between bacteria and their host cells, i.e., adhesion via lectins and sugars. The sugar composition was analysed by light and electron microscopic labelling experiments using the lectins Con A, WGA and SBA. In general, only the posterior body surface ofJoenia which is colonized with bacteria is labelled. The demonstrated sugars are found in fibrous glycocalyx portions surrounding the attachment sites of the bacteria. Such glycocalyx fibres in combination with the electron dense material supporting the attachment sites seem to be the prerequisites for bacterial attachment. InD. glabra, however, a role for sugars in mediating the attachment could not be demonstrated. Removal of the ectobiotes using antibiotics revealed that the specialized contact sites ofJoenia are present in the absence of bacteria and thus possibly serve to attract bacteria. Nothing, however, remains of the former attachment sites in bacteria-freeDevescovina cells. Attachment sites in this case could be induced by bacterial contact. There is not one general mechanism for bacterial attachment to termite flagellates; rather, adhesion seems to follow different strategies.Abbreviations Con A concanavalin A - DAB 3,3-diaminobenzidine tetrahydrochloride - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - FA formaldehyde - FITC fluorescein isothiocyanate - GA glutaraldehyde - PB Soerensen's phosphate buffer - PC phase contrast - pen/strep penicillin and streptomycin - SBA soybean agglutinin - SEM scanning electron microscope - TBS Tris buffer saline - TEM transmission electron microscope - WGA wheat germ agglutinin Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Identification of thigmoresponsive loci for cell differentiation inUromyces germlings

Summary The sites alongUromyces appendiculatus germ tubes that are responsive to topographical induction for appressorium formation were determined using glass micropipettes. The germ tubes were perturbed with the micropipettes at different sites and durations. The most responsive region of the germ tubes for appressorium formation was within 0–10 m from the cell apex where >90% of the perturbed germ tubes developed appressoria. Furthermore, only the cell surface in contact with the substratum was responsive. Appressoria could not be induced to form, under any conditions, by perturbing cell-substratum regions of the germ tubes more than 40 m from the apex. Maximum appressorium formation occurred when the perturbing micropipette was left in place for >20 min.     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Mode of action and antifungal properties of two cold-adapted chitinases

The mode of action of two chitinases from the Antarctic Arthrobacter sp. strain TAD20 on N-acetyl-chitooligomers and chitin polymers has been elucidated. Identification of the length of chitin oligomers following enzymatic hydrolysis was verified by using HPLC-based analysis. It was observed that the length of the oligomer is important for enzyme action. The enzymes cannot effectively hydrolyze chitin oligomers with a degree of polymerization lower than four. ArChiA is an endochitinase which hydrolyzes chitin substrates randomly, whereas ArChiB is an exochitinase which degrades chitin chains and N-acetyl-chitooligomers from the nonreducing end, releasing N-N-diacetyl-chitobiose. ArChiB (100 g/ml) inhibited spore germination and hyphal elongation of the phytopathogenic fungus Botrytis cinerea by 15% and 30%, respectively. A more pronounced effect was observed with ArChiA (100 g/ml) resulting in 70% inhibition of spore germination and 60% inhibition of germ tube elongation. A slight additive effect was observed, when the two enzymes were used in combination, only on the inhibition of germ tube elongation.Communicated by G. Antranikian     

A lectin cytochemical study of glycoconjugates in the human retina

Summary The binding to morphologically normal human retina of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities was studied. Eight formalin-fixed and paraffin-embedded eyes were examined. Photoreceptor cells bound Lens culinaris (LCA), wheat germ (WGA), peanut (PNA) and Ricinus communis (RCAI) agglutinins, and concanavalin A (ConA). The outer segment region was labeled more strongly than the inner segment region, and PNA labeled only cones. All these lectins except PNA bound to both plexiform layers, and all but PNA and RCAI to the nuclear layers. Pretreatment with neuraminidase to remove sialic acid resulted in increased binding of RCAI and PNA, which now labeled both rods and cones, and in decreased binding of WGA. Bandeiraea simplicifolia (BSAI), Dolichos biflorus (DBA), soybean (SBA), Ulex europaeus (UEAI), and Lotus tetragonolobus (LTA) agglutinins, as well as pokeweed mitogen (PWM) reacted only with retinal vascular endothelial cells, which were also labeled with the other lectins. The results indicate that -mannose, -glucose, -galactose, N-acetyl-d-glucosamine and N-acetylneuraminic acid are present in glycoconjugates of human neuroretina.