Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.     

Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambiae gut extracts and haemolymph

Lectins that agglutinate red blood cells (RBC) were demonstrated in Anopheles gambiae mosquito haemolymph and gut extracts. No apparent differences in haemagglutinin titres were detected between male and female mosquitoes and overall agglutinin levels were not increased following a bloodmeal. Titres were highest in the haemolymph and midgut extracts versus human AB, horse, chicken and goat RBCs and in hindgut against human AB, chicken and sheep; foregut extract gave relatively low titres. Adsorption of haemolymph and gut extracts with selected RBCs coupled with carbohydrate inhibition and the use of enzyme-treated RBCs revealed the presence of multiple (hetero-) agglutinins. An.gambiae lectins were specific for (1-1)-, (1-4)- or (1-6)-linked glucose based disaccharides, glucose and its (1-2) or (1-3) linkages with fructose and, to a lesser extent, aminated or N-acetylated glucose, or galactose and its deoxy derivatives. This study presents the first report of the occurrence of heterogenous anti-RBC agglutinins in haemolymph and gut extracts of the mosquito An.gambiae, together with the sugar-binding specificities of these lectins.     

A putative carbohydrate-binding domain of the lactosebindingCytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of thel-fucose-bindingUlex europaeus anti-H(O) lectin

The complete amino acid sequence of a lactose-bindingCytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of thel-fucose-bindingUlex europaeus lectin I (UEA-I).Abbreviations BPA Bauhinia purpurea lectin - Con A concanavalin A - CMA-I Cytisus multiflorus lectin I - CMA-II Cytisus multiflorus lectin II - CSA-I Cytisus sessilifolius lectin I - CSA-II Cytisus sessilifolius lectin II - CSII Cytisus scoparius lectin II - ECorL Erythrina corallodendron lectin - GSIV Griffonia simplicifolia lectin IV - HPLC high performance liquid chromatography - LAA-I Laburnum alpinum lectin I - LAA-II Laburnum alpinum lectin II - LOL Lathyrus ochrus lectin - LTA Lotus tetragonolobus lectin - MAH Maackia amurensis haemagglutinin - PSA Pisum sativum lectin - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid - UEA-I Ulex europaeus lectin I - UEA-II Ulex europaeus lectin II - VFA Vicia faba lectin     

Carbohydrate-binding properties of an immobilized alpha-D-galactopyranosyl-binding protein (lectin) from the seeds of Bandeiraea simplicifolia.

The alpha-D-galactopyranosyl-binding lectin from Bandeiraea simplicifolia has been coupled to cyanogen bromide-activated Sepharose 4B. Using this immobilized system, we have been able to study the interaction of the lectin with model carbohydrate-protein conjugates and polysaccharides, and to reaffirm this protein's carbohydrate-binding specificity. The opportunity for the isolation of biopolymers containing alpha-D-galactopyranosyl end-groups is demonstrated by the single-step purification of a new galactomannan from the seeds of Cassia alata.     

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.     

Mn2+-electron spin resonance spectra of several lectins.

Mn2+-ESR spectra of soybean, wax bean and lima bean agglutinin at Q- and X-band frequencies show nearly axially symmetric zero field splitting (ZFS); the dominant anisotropic term of the spin hamiltonian is the quadratic ZFS interaction. There is a relatively large distribution of ZFS parameters. No effects of specific inhibitor (N-acetylgalactosamine) on the soybean agglutinin spectrum were observed. The stoichiometric complex obtained on addition of Mn2+ to a Mn2+-free sample of this protein has a spectrum similar to that of the native protein. The small changes in the spectrum are interpreted in terms of a wider distribution of the ZFS parameters at the Mn binding site. Addition of Ca2+ to Mn2+-soybean agglutinin sharpens the lines, possibly because Ca2+ increases the rigidity of the complex.     

Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus

Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp³² → Glu³²), rPL-N34S (Asn³⁴ → Ser³⁴) and rPL-V36A (Val³⁶ → Ala³⁶) whereas binding site 2; rPL-D61E (Asp⁶¹ → Glu⁶¹), rPL-N63S (Asn⁶³ → Ser⁶³) and rPL-V65A (Val⁶⁵ → Ala⁶⁵). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.     

Antiradical and anti-H2O2 properties of polyphenolic compounds from an aqueous peppermint extract

Polyphenolic compounds such as eriocitrin, luteolin-7-O-rutinoside, diosmin, hesperidin, narirutin, isorhoifolin, rosmarinic and caffeic acids were identified in an aqueous extract (Ex) obtained from peppermint leaves (Menthae x piperitae folium). The content of polyphenols in Ex was as follows: eriocitrin 38%, luteolin-7-O-rutinoside 3.5%, hesperidin 2.9%, diosmin 0.8%, isorhoifolin 0.6%, narirutin 0.3%, rosmarinic acid 3.7% and caffeic acid 0.05%. The strongest antiradical activity (determined as DPPH* scavenging features) was observed for luteolin-7-O-rutinoside, eriocitrin and rosmarinic acid. Caffeic acid and hesperidin revealed a lower antiradical activity while isorhoifolin, narirutin and diosmin showed the lowest activity. The strongest anti-H2O2 activity was observed for eriocitrin, a little lower for rosmarinic acid. The rate of hydrogen peroxide scavenging activity displayed by luteolin-7-O-rutinoside and caffeic acid was lower than that of rosmarinic acids. Hesperidin appeared to be a very weak scavenger of hydrogen peroxide. Almost no anti-H2O2 activity was demonstrated for diosmin, narirutin and isorhoifolin. Among examined flavonoids, the strongest antiradical and anti-H2O2 activity was shown for compounds with two hydroxy groups bound to the Bring in ortho position in relation to each other. Replacement of one hydroxy group in the Bring with a methoxy group or removing one hydroxy group leads to decrease of antiradical and anti-H2O2 activity of flavonoids. Our results suggest that eriocitrin is a powerful peppermint antioxidant and a free radical scavenger.     

Stimulation by iodide of H(2)O(2) generation in thyroid slices from several species

The regulation of thyroid metabolism by iodide involves numerous inhibitory effects. However, in unstimulated dog thyroid slices, a small inconstant stimulatory effect of iodide on H(2)O(2) generation is observed. The only other stimulatory effect reported with iodide is on [1-(14)C]glucose oxidation, i.e., on the pentose phosphate pathway. Because we have recently demonstrated that the pentose phosphate pathway is controlled by H(2)O(2) generation, we study here the effect of iodide on basal H(2)O(2) generation in thyroid slices from several species. Our data show that in sheep, pig, bovine, and to a lesser extent dog thyroid, iodide had a stimulatory effect on H(2)O(2) generation. In horse and human thyroid, an inconstant effect was observed. We demonstrate in dogs that the stimulatory effect of iodide is greater in thyroids deprived of iodide, raising the possibility that differences in thyroid iodide pool may account, at least in part, for the differences between the different species studied. This represents the first demonstration of an activation by iodide of a specialized thyroid function. In comparison with conditions in which an inhibitory effect of iodide on H(2)O(2) generation is observed, the stimulating effect was observed for lower concentrations and for a shorter incubation time with iodide. Such a dual control of H(2)O(2) generation by iodide has the physiological interest of promoting an efficient oxidation of iodide when the substrate is provided to a deficient gland and of avoiding excessive oxidation of iodide and thus synthesis of thyroid hormones when it is in excess. The activation of H(2)O(2) generation may also explain the well described toxic effect of acute administration of iodide on iodine-depleted thyroids.     

Isolation and structure of several peptides from porcine hypothalami

Ten peptides were isolated from porcine hypothalami and structurally elucidated. These included four dipeptides Arg-Phe, Phe-Tyr, Val-Trp, and Tyr-Phe; a tripeptide Lys-Phe-Tyr; two tetrapeptides Gly-Lys-Val-Asn and Phe-Glu-His-Glu, a nonapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; a decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe and a hexadecapeptide Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe-Asn-Leu. The tetrapeptide Gly-Lys-Val-Asn, the nonapeptide, the decapeptide and the hexadecapeptide most probably represent artifact fragments of alpha- and beta-chains of porcine hemoglobin. The natural or synthetic Gly-Lys-Val-Asn and Phe-Glu-His-Glu had some growth hormone releasing activity while Val-Trp, Tyr-Phe and Lys-Phe-Tyr had slight prolactin releasing activity. The biological activities of other peptides have not been determined yet.     

Isolation and characterization of lectins from kidney beans (Phaseolus vulgaris)

The bioactive properties of lectins obtained from raw and canned red kidney bean (Phaseolus vulgaris) were studied to determine the changes in their bioactivity during the canning process. Phytohaemagglutinin (PHA) was extracted using Affi-gel Blue gel and thyroglobulin-Sepharose and had a molecular weight of 32 kDa. Both the raw and the canned kidney beans possessed the ability to agglutinate red blood cells and inhibit α-glucosidase. The activity found in the canned beans was similar to that from the in the raw kidney beans. However, the amount of lectin that could be extracted from thyroglobulin-Sepharose was much less in the canned samples than in the raw kidney bean samples. The extracted lectin from the raw kidney beans was also subjected to a heating and cooling treatment using a differential scanning calorimeter. The lectin had a nonset denaturation temperature of 77.76 °C and it did not renature upon cooling. In this study, we demonstrated that extracts from raw red kidney bean and canned red kidney bean contain bioactive compounds capable of inhibiting HIV-1 RT in vitro.     

Role of lectins (and rhizobial exopolysaccharides) in legume nodulation.

The lectin recognition hypothesis proposes that plant lectins mediate specificity in the Rhizobium-legume symbiosis. Although the hypothesis was developed eight years before nod genes were identified in rhizobia and sixteen years before Nod factor was shown to be a major determinant of host specificity, experiments performed recently using transgenic lectin plants support its main tenets.     

A comparative study of bark lectins from three elderberry (Sambucus) species

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.     

Bioactive constituents from Dracocephalum subcapitatum (O. Kuntze) Lipsky

From an EtOAc extract of Dracocephalum subcapitatum, five flavonoids, calycopterin, xanthomicrol, isokaempferide, luteolin and apigenin, together with five terpenoids, oleanolic acid, ursolic acid, geranial, neral and limonene-10-al, were isolated. Among them, citral and limonene-10-al were the most effective components against epimastigotes of Trypanosoma cruzi, the parasitic agent of Chagas disease.     

Studies on hemagglutinins (lectins) from plasma of murrel fish, family Channidae

Hemagglutinating activity can be identified in the plasma of different species of murrel fish. This activity may be divided into four types according to their agglutinability towards erythrocytes from different sources. Type I plasma agglutinates human blood group A erythrocytes, type II can agglutinate neuraminidase treated human A B O erythrocytes, type III shows no agglutinating activity towards human erythrocytes, while type IV agglutinates human erythrocytes non-specifically. All of them bind to DEAE-cellulose but elute out by different salt concentrations. Type IV plasma is found to be a combination of three separate hemagglutinins, which are separable by sequential binding to human A B O erythrocytes. Blood group A specific lectin activity is purified from this plasma using formalinised A group erythrocytes. The apparent homogeneity of this purified lectin is established by polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion. This agglutinin is antigenically identical with that isolated from type I plasma by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose column. Their molecular weights are also found to be identical (Mr 140,000) in polyacrylamide gel electrophoresis, having two identical subunits. Forssman glycolipid (0.03 mM) was found to be the most potent inhibitor of agglutination, although Gal beta 1-3 GalNAc (0.09 mM) is also a good inhibitor. Exhaustive dialysis of the purified lectin (hemagglutinin) against EDTA denatures it irreversibly by dissociating it to its subunit structure. Thus human A group agglutinating activity isolated from type I and type IV plasma are identical.     

Tropane alkaloids from Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae)

Six tropane alkaloids were isolated from the Sri Lankan endemic plant Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae) and structurally elucidated by NMR and MS measurements. Three of them, erythrozeylanines A [1R,3R,5S,6R-6-acetoxy-3-(3',4',5'-trimethoxybenzoyloxy)tropane], B [cis-3 beta-(cinnamoyloxy)tropane], and C [cis-6 beta-acetoxy-3 alpha-(cinnamoyloxy)tropane] are new, whereas the others have already been found in other Erythroxylum species. For the first time, the absolute configuration of a tropane alkaloid (erythrozeylanine A) has been determined by quantum chemical CD calculations.     

Purification and physico-chemical properties of lectins from the jack fruit (Artocarpus iutegrifolia)

A haemagglutinating material was isolated and purified from the phosphate buffered saline extract of the seeds of the jack fruit using an immobilized N-aeetyl-D-galactosamine column. This material was composed of two iso-lectins of molecular masses 11 500 and 15 000. The lectins agglutinated native washed red blood cells of the human A, B and 0 groups and sheep, rabbit and mouse erythrocytes. The lectins were found to be composed of single polypeptide chains and they contained no covalently linked sugars. The lower molecular mass material was present in considerably greater quantity than the higher molecular mass component. On isoelectric focussing on PAG the lectins gave a spread of components with calculated pI between 6.0 and 8.3.