Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.).

The lectin of the Indian bean or lablab (Dolichos lablab L.) was purified by affinity chromatography on two types of affinity carriers: O-alpha-D-mannopyranosyl-Separon and Separon-bound ovomucoid. The lectin is homogeneous in the ultracentrifuge: S20, w = 6.14 S, Mr = 110 000; the molecule appears to comprise two pairs of two types of subunits (Mr 16 000 and 40 000), and contains 2% neutral sugar and 0.2 Mn and 0.5 Zn atom respectively. The lectin agglutinates human erythrocytes non-specifically with regard to ABO grouping at a limit concentration of 8 micrograms/ml, and this activity is inhibited most effectively by N-acetyl-D-glucosamine, methyl alpha-D-mannopyranoside and ovomucoid, but not by free D-mannose.     

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

Toxic and antigrowth effects of raw and processed field bean (Dolichos lablab) on albino rats

Samples of freeze dried green field bean (Dolichos lablab) and dry mature bean, were subjected to the following processing methods—heat processing, extraction with 80% ethanol, hexane or dilute acid, protein isolation; and these samples were evaluated for growth promoting value and toxicity. Extraction with 80% ethanol or with dilute acid increased survival period of the animals; but these did not promote growth. Heat processing was essential to destroy antinutritional factors and promote growth. Extraction of the beans with 80% ethanol did not however alter the trypsin inhibitor or haemagglutinin activities. The protein isolate and acid-extracted residue which had low trypsin inhibitor and haemagglutinin activities, did not also promote growth. Thus the trypsin inhibtor and haemagglutinin activities did not completely account for the toxicity to albino rats. However, heat processing of ethanol extracted bean flour indicated that the beneficial effect of ethanol extraction was not apparent, once the samples were heat processed. Dry mature bean dhal was more toxic than the whole bean either dry or green. Supplementation of heat processed field bean with methionine and tryptophan promoted good growth of albino rats and significantly increased the protein efficiency ratio. Part of the Ph.D. thesis entitled “Studies on the factors affecting the nutritive value of field beanDolichos lablab”, University of Mysore, 1977.     

侵染扁豆的三叶草黄脉病毒的鉴定

1987年从泰安表现系统花叶的扁豆上分离到一个分离物B_2,汁液摩擦接种9科38种植物,它可侵染5科10种植物,在扁豆和昆诺藜上引起系统花叶,在苋色藜上引起局部枯斑。该分离物钝化温度为60—65℃,稀释限点为10~(-3)—10~(-4),体外存活期为3—5天。可由桃蚜传播;病毒粒体线条状,大小为700—760×12nm病叶细胞内有风轮状内含体,该分离物与三叶草黄脉病毒的抗血清有明显的-阳性反应。根据这些特性,该病毒属于马铃薯丫病毒组的三叶草黄脉病毒。     

The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack.     

The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂SO₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K_a’s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16 ± 0.002, 0.21 ± 0.004 and 8.45 ± 0.78 (×10^?3) M^?1 respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding.     

Purification and Separation of Eight Steroidal Plant-growth Regulators from Dolichos lablab Seed

Purification and separation of eight steroidal plant-growth regulators, dolicholide, dolichosterone, homodolicholide, homodolichosterone, brassinolide, castasterone, 6-deoxocastasterone and 6-deoxodolichosterone, from immature seed of Dolichos lablab were accomplished using various chromatographic techniques. The reversed-phase HPLC of a number of steroidal plant growth regulators was also studied.     

Purification and Properties of Trypsin Inhibitor from Hakuhenzu Bean (Dolichos lablab)

A highly purified trypsin inhibitor was obtained from the oriental plant Hakuhenzu bean (Dolichos lablab) by column chromatography on DEAE-Sephadex and gel-filtration on Sephadex G–75. The purified Hakuhenzu bean trypsin inhibitor (HTI) was obtained as a chemically homogeneous protein, and was stable to heat and to enzymes such as pepsin. It shows no obvious maximum at 280 nm in the ultraviolet absorption spectrum, and it contains more than 20% carbohydrate as galactose and 10% hexosamine as glucosamine. The molecular weight of this inhibitor was determined to be approximately 9,500 by gel-filtration. The protein contained 59 residures of amino acids; Lys₃, His₄, Arg₁, Asp₈, Thr₃, Ser₉, Glu₆, Pro₅, Gly₁, Ala₃, l/2Cys₁₀, Val₁, Ile₁, Leu₂, Tyr₁, Phe₁, from which a molecular weight of 6,400 is obtained. No methionine and tryptophan were found in the amino acid composition of the inhibitor. This inhibitor showed inhibitory activity against α-chymotrypsin in addition to trypsin.     

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.     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Effects of Growth Retardants on the Growth of Excised Roots of Dolichos lablab L. in Culture

The effects of various growth-retarding chemicals on the growthof excised roots of Dolichos lablab in sterile culture are investigated.Application of a range of concentrations of CCC, phosfon, andB-995 inhibited growth in length of the roots by reducing thefrequency of cell division, with little or no effect on cellelongation. Treated roots had generally lower DNA, RNA, andprotein contents than controls, although CCC-treatment significantlyenhanced the soluble nitrogen content of the roots. The inhibitoryeffects of growth retardants were not reversed by IAA or GA,but substances like choline chloride and pyridoxine hydrochloridewere partially effective in reinstating normal growth in rootsgrowing in concentrations of CCC producing about 50 percentinhibition of growth in length.     

An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC-MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.     

Nodulation and growth ofLablab purpureus (Dolichos lablab) in relation to rhizobium strain,liming and phosphorus

Summary Greenhouse experiments were done with two purposes: (1) to identify strains of rhizobia effective and acid-tolerant in symbiosis withLablab purpureus, and (2) to determine whether soil acidity or the symbiotic condition increased the phosphate requirement for growth.Five rhizobial strains were tested in one neutral soil, two acid soils, and the two acid soils limed to pH 6.6. In the neutral and limed soils, three of the strains were effective (CB1024, CB756, TAL169), but only two strains (CB756, TAL169) remained effective in acid soil.Strain CB756 and plus-N treatments were further compared in a factorial trial involving combinations of five levels of P with lime, no lime and CaCl₂ treatments, applied to an acid soil. Some of the treatments were also applied to plants inoculated with CB1024. Between the N-fertilized and CB756 treatments there was no clear difference in growth response to applied P, and the critical internal concentration of P for 95% of maximal growth was the same (0.22% shoot dry weight). Increasing P beyond levels needed for maximal growth increased nodulation and N concentration in plants inoculated with CB756. It lowered N concentration in N-fertilized plants. There was evidence suggesting that the P requirement of symbiotic plants increased if the soil was acid, or if CB756 were replaced by CB1024 as microsymbiont; but the critical statistical interactions were not significant.     

Host Status of Crotalaria juncea,Sesamum indicum,Dolichos lablab,and Elymus glaucus to Meloidogyne javanica

Reproduction of Meloidogyne javanica on Crotalaria juncea PI 207657 and cv. Tropic Sun, Sesamum indicum, Dolichos lablab, and Elymus glaucus was assessed using a root-gall index, a reproductive index obtained by dividing the final population of juveniles (J2) in soil by the initial J2 population (Pf/Pi), and the number of J2 per gram of root recovered from roots by mist chamber extraction. Lycopersicon esculentum (cv. UC 204 C) was included as a susceptible host. The root-gall index and soil reproductive index were poor indicators of the host status of our test plants as compared with mist chamber extraction of J2 from roots. Lycopersicon esculentum had a mean root-gall index of 7.8. Some plants of S. indicum and E. glaucus had a few galls and other plants had none, with mean root-gall indices of 1.6 and 0.8, respectively. No galls were observed in C. juncea and D. lablab. Lycopersicon esculentum had the highest mean soil Pf/Pi value (mean = 1.93), while in C. juncea and some replicates of S. indicum no soil J2 were found. Even though some replicates had no galls, all replicates supported nematode reproduction. The mean numbers of J2 per gram root after 5 days of mist extraction were 447.7, 223.3, 165.5, 96.9, 42.3, and 41.9 for D. lablab, L. esculentum, E. glaucus, S. indicum, and C. juncea PI 207657 and cv. Tropic Sun, respectively. Accurate assessment of nematode resistance was influenced by sampling time and the nematode extraction technique used. Individual plants of both C. juncea and S. indicum supported nematode reproduction to some extent; however, both C. juncea and S. indicum have potential as cover crops to reduce M. javanica numbers.     

Effect of in vivo administered plant growth hormones on the development of amylase and protease during germination of indian bean (Dolichos lablab L. var. lignosus) seeds

The plant growth hormones — BA, GA₃ or IAA (alone or in combination) in single dose were injected into the centre of intact imbibed seed, germinated in the presence of water and monitored for root length, shoot length, and activities of α-amylase and proteases during a 10-day germination period of Indian bean seeds. A significant increase was noticed in the root length and shoot length compared to the respective controls. Injection of BA, GA₃ or IAA alone resulted in a significant increase in the activity of α-amylase. The increase in the activities of proteases (acidic, neutral and alkaline) was not affected with either BA or GA₃ during the early stage of germination, but the fall in their activities in the later stages of germination was suppressed. However, in vivo administration of IAA alone or in combination with BA or GA₃ resulted in faster and higher development of α-amylase and protease(s) activities. These results indicated that more than one hormone is necessary for inducing the development of α-amylase and proteases at early stages, and also responsible for maintaining the higher activities in the later stages of germination.     

Tryptophan environment, secondary structure and thermal unfolding of the galactose-specific seed lectin from Dolichos lablab: fluorescence and circular dichroism spectroscopic studies

Fluorescence and circular dichroism spectroscopic studies were carried out on the galactose-specific lectin from Dolichos lablab seeds (DLL-II). The microenvironment of the tryptophan residues in the lectin under native and denaturing conditions were investigated by quenching of the intrinsic fluorescence of the protein by a neutral quencher (acrylamide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The results obtained indicate that the tryptophan residues of DLL-II are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains residing close to at least some of the tryptophan residues under the experimental conditions. Analysis of the far UV CD spectrum of DLL-II revealed that the secondary structure of the lectin consists of 57% alpha-helix, 21% beta-sheet, 7% beta-turns and 15% unordered structures. Carbohydrate binding did not significantly alter the secondary and tertiary structures of the lectin. Thermal unfolding of DLL-II, investigated by monitoring CD signals, showed a sharp transition around 75 degrees C both in the far UV region (205 nm) and the near UV region (289 nm), which shifted to ca. 77-78 degrees C in the presence of 0.1 M methyl-beta-D-galactopyranoside, indicating that ligand binding leads to a moderate stabilization of the lectin structure.