Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Purification and Characterization of Proteins from the 2S Fraction from Seeds of the Brassicaceae Family

The 2S protein fractions from Brassica rapa, Brassica oleracea,and Brassica napus seeds have been obtained and their componentspurified and characterized. These albumins represent about 13%of the total seed protein extracted with saline buffer. Thecalculated molecular weights of the eleven purified proteinsare very similar, about 14 500 Daltons, although two componentsisolated from B. napus exhibit a lower molecular weight. Theamino acid compositions of the isolated proteins present a seriesof common features: a high content of Cys and basic residuesas well as a very high amount of Pro (15%) and Glx (30%). Theeleven purified proteins cross-react with antibodies againstSin a I, the 2S protein from yellow mustard seeds. The obtainedresults suggest the existence of homology between the 2S albuminsof Brassicaceae seeds. Key words: Brassicaceae, seeds, storage protein     

Purification and characterization of bovine mannan-binding protein

Bovine mannan-binding protein (bMBP) was observed in serum byits Ca²⁺ -dependent binding to mannan and by an M_rof 28 kDaunder reducing conditions on sodium dodecyl sulphate—polyacrylamidegel electrophoresis (SDS—PAGE). The lectin was isolatedby precipitation with polyethyl-eneglycol (PEG), affinity chromatographyon mannan—Sepharose eluted with EDTA, and absorption onSepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies.Fractions containing the lectin were reapplied to mannan—Sepharoseand eluted first with N-acetyl-D-glucosamine (GlcNAc) to removeconglutinin, and then with mannose to elute the 28 kDa lectin.Further purification was achieved by ion-exchange chromatographyon Mono-Q and by man-nose-gradient elution from a mannan-Sepharosecolumn. SDS—PAGE of the purified lectin showed three highmolecular weight bands under non-reducing conditions. The reducedprotein gave a single band of 28 kDa. On gel permeation chromatographyunder non-dissociating conditions, the protein emerged at avolume corresponding to M_r     

The Lectins of Sophora japonica: I. Purification Properties and N-Terminal Amino Acid Sequences of Two Lectins from Leaves

Two lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like the Sophora seed lectin, LLI and LLII are tetrameric glycoproteins containing a single subunit with respect to size. The subunits of LLI (32 kilodaltons) and LLII (34 kilodaltons) are slightly larger than those of the seed lectin (29.5 kilodaltons). The three Sophora lectins display indistinguishable specificities, amino acid compositions, specific hemagglutinin activities, and extinction coefficients. Although very closely related to the seed lectin, the leaf and seed lectins are not immunologically identical and they differ in subunit molecular weights, carbohydrate content, and in the pH sensitivity of their hemagglutinin activities. N-terminal amino acid sequence analysis shows that although they are homologous proteins, the three Sophora lectins are products of distinct genes.     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Molecular properties of the partially SDS-resistant lectin from the albumen gland of Helix pomatia and demonstration of lectin-related molecules on the surface of H. pomatia haemocytes

Lectins (agglutinins) are components of the immunobiologicalrecognition system of vertebrates and invertebrates. The presentstudy focused on the molecular properties of the agglutininfrom the albumen gland of Helix pomatia (HPA) and on the occurrenceof lectin-related molecules on the surface of H. pomatia haemocytes.According to the current model (Hammarström et al., 1972,Scandinavian Journal of Immunology, 1: 259–301), the hexamericHPA of about 79 kDa is composed of three non-covalently associateddimers (26 kDa), each consisting of two disulphide-bridged 13kDa monomers. However, on native-gradient polyacrylamide gelelectrophoresis (PAGE), we obtained high molecular weight bandsrepresenting lectin polymers. The stepwise dissociation of thesewas achieved by incubation with SDS at temperatures from 20to 40°C (1 h) and at 100°C (10 min). The results obtainedon SDS–PAGE included the occurrence of partially SDS-resistanthexamers of about 66 kDa, of two dimer bands of 22 and 19 kDa,and of two minor heteromonomer fractions. Complete dissociationinto heteromonomers of 13 and 11 kDa was achieved by boilingthe lectin (10 min) with SDS under reducing conditions. Fornative lectin molecules, both monomers occurred as disulphide-linkedhomodimers. Monomers or dimers electroeluted from an SDS–gel,reassociated to SDS-resistant oligomers upon re-electrophoresis.Finally, molecules antigenetically related to the lectin wereextracted from the membrane of H. pomatia haemocytes. Anti-HPAantibodies recognized peptides with an apparent molecular weightof about 30 and 56 kDa, which were shown to represent cell-surfacemolecules. (Received 4 March 2008; accepted 9 September 2008)     

Ginkgo biloba Expresses Calreticulin,the Major Calcium-Binding Reticuloplasmin in Eukaryotic Cells

Calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was characterized in Ginkgo biloba L. pollen and seeds. Electrophoretic analysis of the partly purified extracts showed the presence of two protein bands of 57 and 50kDa apparent molecular masses, which strongly cross-reacted with antibodies against plant calreticulins. Amino acid sequence comparison with other plant and animal calreticulins revealed a much higher similarity of the N-terminus of Ginkgo calreticulins with the homologue from angiosperms rather than with that from mammals. The finding of calreticulin in Ginkgo is indicative of the conservation also in gymnosperms of Ca²⁺ homeostatic mechanisms, which seem to rely on the same molecular components as all eukaryotic cells.     

Affinity purification, physicochemical and immunological characterization of a galactose-specific lectin from the seeds of Dolichos lablab (Indian lablab beans)

A galactose-specific lectin has earlier been isolated from the seeds of Dolichos lablab in our laboratory by conventional protein purification methods. We now established conditions to bind the lectin on Sepharose-galactose gel in the presence of 1.5 M ammonium sulfate in Tris-buffered saline, pH 7.4. It can be specifically eluted with 0.3 M galactose. The purified lectin is a glycoprotein, binds to Con A, agglutinates erythrocytes, and has an apparent native molecular weight of 120 +/- 5 kDa. In SDS-PAGE under reducing conditions, it dissociates into two subunits of molecular mass (Mr) 31 and 29 kDa. Among a number of sugars tested for inhibitory activity of the lectin, galactose was found to be a potent inhibitor. Rabbit polyclonal antibody to the purified lectin specifically reacted with the lectin subunits in Western blot analysis and additionally, an antibody raised to the isolated 31 kDa subunit show reactivity with both the subunits. Amino terminal sequences of both the subunits are identical. The purified lectin is stable up to 40 degrees C with a pH optimum of 7.4. The lectin has a high content of acidic amino acids and lacks sulfur-containing amino acids. Chemical modification of the lectin with group-specific reagents indicates the possible role of histidine, lysine, and tyrosine residues in lectin activity.     

A monomeric protein with hemagglutinating activity from seeds of Vigna mungo (Phaseolus mungo).

Black gram (Vigna mungo) seeds are shown to contain a lectin with certain unusual features. The lectin agglutinates only trypsinized red cells, and its sugar specificity is complex as none of the common sugars, oligosaccharides or complex polysaccharides exhibit any affinity for the lectin. The purified lectin has a molecular weight of 58 kDa and is a monomer. Unlike other plant lectins, antibodies to the P. mungo lectin do not exhibit any immunological cross reactivity. The clot forming ability of the lectin is unusual in that the clot once formed is rapidly disaggregated indicated that it induces, as yet undefined, certain membrane alterations.     

Characterization of HSP-70 cognate proteins from wheat

Summary Animal and plant cells contain a family of constitutively expressed HSP-70 cognate proteins that are localized in different subcellular locations and are presumed to play a role in protein folding and transport. Utilizing antibodies raised against the yeast endoplasmicreticulum-localized HSP-70 cognate termed BiP/GRP-78, as well as antibodies raised against the Escherichia coli HSP-70 protein DnaK, we have identified and characterized a large family of closely related proteins in wheat. One protein band of 78 kDa that is apparently closely related to yeast BiP was localized in the endoplasmic reticulum. This band cross-reacted with the yeast BiP but not with the DnaK-specific antibodies. The yeast BiP antibodies also recognized a cytoplasmic protein of 70 kDa that is probably related to the HSC-70 cognate proteins. These two proteins were further confirmed as HSP-70 cognates by their ability to bind to an ATP-agarose column. Probing of proteins from purified wheat mitochondrial preparations with the yeast BiP and DnaK-specific antibodies showed that this organelle contained a family of HSP-70-related proteins. The yeast BiP antibodies recognized two mitochondrial proteins of 60 and 58 kDa, but failed to detect any protein in the size rang of 70 to 80 kDa. However, the presence of immunologically distinct proteins of 90 and 78 kDa, as well as of lower molecular weight from this family in the mitochondria, was shown by probing with the DnaK-specific antibodies. A new protein of 30 kDa, cross-reacting with anti-yeast BiP antibodies, was detected only in developing seeds, close to their maturity. The evolution of HSP-70 cognate proteins in wheat as shown in this study is discussed.     

Changes in patterns of protein synthesis during haustorial development of Striga hermonthica (Del.) Benth. seedlings

This study focuses upon the developmental transition of theparasitic plant Striga hermonthica from its freeliving state(germinated seedling) to its parasitic state after developmentof an infection organ: the haustorium. A new method has beendeveloped that allows the production of gram quantities of germinatedand haustorially-induced Striga seedlings, thereby facilitatingbiochemical and molecular analysis of haustorial induction.Water-soluble proteins have been extracted from germinated seeds(stage A) and seedlings treated with 2,6-dimethoxy-p-benzoquinone(2,6-DMBQ) to induce haustorium (stage B). Samples were analysedby two-dimensional polyacrylamide gel electrophoresis and quantitativeas well as qualitative differences could be observed. In particulara group of four highly abundant acidic proteins (molecular weight39 kDa, pl 5.1, 5.3, 5.3, 5.6) and three other proteins (molecularweight 12 kDa, pl 6.9; 17 kDa, pl 4.4; 17 kDa, pl 4.45) wereseen in stage A while at least four proteins (molecular weight21.5 kDa, pl 6.4; 21.5 kDa, pl 6.3; 31 kDa, pl 5.1; 34 kDa,pl 6.2) were present in greater abundance in stage B. In orderto compare watersoluble protein with newly synthesized proteinpatterns, mRNAs from the two stages of development were isolatedand cell-free translation products analysed by 2-D PAGE. Two-Dgels of cell-free translation products showed the appearanceof six proteins in stage B (molecular weight ranging from 10to 35 kDa) and the presence of three acidic proteins in stageA with one protein (molecular weight 40 kDa) very similar insize to the triplet of proteins in the water-soluble protein2-D gels. Key words: Striga hermonthica (Del.) Benth., haustorium, 2-D PAGE, 2,6-DMBQ, translation in vitro     

Elderberry (Sambucus nigra L.) seed proteins inhibit protein synthesis and display strong immunoreactivity with rabbit polyclonal antibodies raised against the type 2 ribosome-inactivating protein nigrin b

Affinity chromatography-purifled elderberry (Sambucus nigraL.) seed proteins strongly inhibited protein synthesis and displayedthe 28S rRNA N-glycosidase activity characteristic of all typesof ribosome-inactivating proteins (RIPs). Western blot analysisrevealed several proteins that reacted with antibodies raisedagainst the novel non-toxic type 2 ribosome-inactivating proteinnigrin b isolated from elder bark, thus indicating the presenceof a new type 2 RIP. Key words: Anti-nigrin b antibodies, protein synthesis, seeds, elder seeds, Sambucus nigra     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Purification and Partial Characterization of a Lectin from the Bark of Japanese Elderberry (Sambucus sieboldiana)

A lectin [Sambucus sieboldiana agglutinin (SSA)] was purifiedfrom the twigs of Sambucus sieboldiana by repeated affinitychromatography on fetuin-Sepharose. SSA had a molecular weight(Mr) of approximately 160 K on gel filtration and consistedof two types of subunit of which the molecular weights rangedfrom 31 to 37 K. SSA agglutinated human erythrocytes irrespectiveof their blood type and the hemagglutination was inhibited bya very low concentration of Neu5Ac(2-6)lactose, suggesting thatSSA has a carbohydrate-binding specificity similar to that ofthe lectin previously isolated from the bark of S. nigra (SNA).However, the Ouchterlony double-diffusion analysis of theselectins with an antibody raised against SSA showed that SSAwas immunologjcally related but not identical to SNA. (Received January 17, 1989; Accepted June 19, 1989)     

Binding-site specificity of lectins from Bauhinia purpurea alba,Sophora japonica,and Wistaria floribunda

The binding-site specificities of lectins isolated from the seeds of Baihinia purpurea alba, Sophora japonica, and Wistaria floribunda were studied by hemagglutination-inhibition assays utilizing a variety of saccharides as inhibitors. For Bauhinia lectin, 2-acetamido-2-deoxy-d-galactose was found to be the best monosaccharide inhibitor and the free monosaccharide inhibitor was as active as its glycosides. d-Galactose was a weak inhibitor and so were some of its glycosides. Some of the oligosaccharides having a d-galactose nonreducing terminus were good inhibitors, but substitution on the d-galactose or 2-acetamido-2-deoxy-d-galactose residues with other saccharides abolished the inhibitory activity. No specificity for anomeric configuration or linkage position could be demonstrated. The presence of aromatic aglycon groups did not enhance inhibitory activity of the saccharides tested and, in some cases, the inhibitory activity was decreased. In contrast to the results for the Bauhinia lectin, compounds having aromatic aglycon groups were markedly better inhibitors for Sophora and Wistaria lectins than the corresponding compounds without aromatic aglycons. d-Galactose was a weak inhibitor for Sophora and Wistaria lectins, whereas 2-acetamido-d-galactose was a poor inhibitor of Sophora lectin but a good inhibitor of Wistaria lectin. Sophora and Wistaria lectins were somewhat similar in their activity as some of the saccharides having a d-galactose in penultimate position to an l-fucose residue were weak inhibitors. However, Sophora lectin has a binding preference for β anomers, whereas Wistaria lectin did not demonstrate a clear preference for α or β anomers. For some pairs of compounds, the α was a better inhibitor than, the β anomer; in other cases, the reverse was true.     

Purification and characterization of a mannose/N-acetyl-D-glucosamine-specific lectin from the seeds of Platymiscium floribundum Vogel

Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins.     

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)⁺ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)⁺ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.     

CuZn-Superoxide Dismutases from the Fern Equisetum arvense and the Green Alga Spirogyra sp.: Occurrence of Chloroplast and Cytosol Types of Enzyme

CuZn-superoxide dismutase (SOD) from horsetail (Equisetum arvense)was purified to a crystalline state and that from pond scum(Spirogyra sp.) was purified to a mixture of three isozymes.The purified CuZn-SODs from the fern and the green alga showsimilar properties to those of the angiosperm and mammalianenzymes with respect to molecular weight, subunit structure,absorption spectrum, circular dichroism spectrum and the effectof modification of the arginine residues by 2,3-butanedioneon activity. Horsetail and pond scum contained three isozymeseach of CuZn-SOD. These isozymes are divided to two types: onetype gave a cross-reaction with antibody raised against chloroplast-typeCuZn-SOD from spinach and other type cross-reacted with antibodyraised against cytosol-type CuZn-SOD from spinach. Thus, itappears that the divergence of the chloroplast and cytosol typesof CuZn-SOD started at a very early stage in the molecular evolutionof this enzyme. (Received January 30, 1989; Accepted April 19, 1989)     

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.